Multiple-tumor aberrant growth genes

ABSTRACT

The present invention relates to the multi-tumor Aberrant Growth (MAG) gene having the nucleotide sequence of any one of the strands of any one of the members of the High Mobility Group protein genes or LIM protein genes, including modified versions thereof. The gene and its derivatives may be used in various diagnostic and therapeutic applications.

This application is a 371 of International Patent Application No.PCT/EP96/00716, filed Feb. 19, 1996, which claims priority fromapplication EPO 9521951.1, Jul. 14, 1995 and application EPO 95200390.3,filed Feb. 17, 1995.

The present invention relates to the identification of the High MobilityGroup (HMG) protein gene family as a family of genes frequentlyassociated with aberrant cell growth as found in a variety of bothbenign and malignant tumors. The invention in particular relates to theidentification of a member of the HMG gene family as the broadly actingchromosome 12 breakpoint region gene involved in a number of tumors,including but not limited to the mesenchymal tumors hamartomas (e.g.breast and lung), lipomas, pleomorphic salivary gland adenomas, uterineleiomyomas, angiomyxomas, fibroadenomas of the breast, polyps of theendometrium, atherosclerotic plaques, and other benign tumors as well asvarious malignant tumors, including but not limited to sarcomas (e.g.rhabdomyosarcoma, osteosarcoma) and carcinomas (e.g. of breast, lung,skin, thyroid), as well as leukemias and lymphomas. The invention alsorelates to another member of the HMG gene family that was found to beimplicated in breaks in chromosome 6.

Furthermore, the invention concerns the identification of members of theLIM protein family as another type of tumor-type specific breakpointregion genes and frequent fusion partners of the HMG genes in thesetumors. The LPP (Lipoma-Preferred Partner) gene of this family is foundto be specific for lipomas. The invention relates in particular to theuse of the members of the HMG and LIM gene family and their derivativesin diagnosis and therapy.

Multiple independent cytogenetic studies have firmly implicated regionq13-q15 of chromosome 12 in a variety of benign and malignant solidtumor types. Among benign solid tumors, involvement of 12q13-q15 isfrequently observed in benign adipose tissue tumors [1], uterineleiomyomas [2, 3], and pleomorphic adenomas of the salivary glands [4,5]. Involvement of the same region has also been reported forendometrial polyps [6, 7] for hemangio-pericytoma [8], and forchondromatous tumors [9, 10, 11, 12]. Recently, the involvement ofchromosome 12q13-q15 was reported in pulmonary chondroid hamartoma [13,14]. Finally, several case reports of solid tumors with involvement ofchromosome region 12q13-q15 have been published; e.g. tumors of thebreast [15, 16), diffuse astrocytomas [17], and a giant-cell tumor ofthe bone [18]. Malignant tumor types with recurrent aberrations in12q13-q15 include myxoid liposarcoma [19], soft tissue clear-cellsarcoma [20, 21, 22], and a subgroup of rhabdomyosarcoma [23].

Although these studies indicated that the same cytogenetic region ofchromosome 12 is often involved in chromosome aberrations, liketranslocations, in these solid tumors, the precise nature of thechromosome 12 breakpoints in the various tumors is still not known.Neither was it established which genes are affected directly by thetranslocations.

In previous physical mapping studies [39], the chromosome 12qbreakpoints in lipoma, pleomorphic salivary gland adenoma, and uterineleiomyoma were mapped between locus D12S8 and the CHOP gene and it wasshown that D12S8 is located distal to CHOP. Recently, it was also foundby FISH analysis that the chromosome 12q breakpoints in a hamartoma ofthe breast, an angiomyxoma and multiple pulmonary chondroid hamartomasare mapping within this DNA interval. In an effort to molecularly clonethe genes affected by the chromosome 12q13-q15 aberrations in thevarious tumors, the present inventors chose directional chromosomewalking as a structural approach to define the DNA region encompassingthese breakpoints.

As a starting point for chromosome walking, locus D12S8 was used. Duringthese walking studies, it was shown that the chromosomal breakpoints aspresent in a number of uterine leiomyoma-derived cell lines areclustered within a 445 kb chromosomal segment which has been designatedUterine Leiomyoma Cluster Region on chromosome 12 (ULCR12) [24].Subsequently, it was found that a 1.7 Mb region on chromosome 12encompasses the chromosome 12 breakpoints of uterine leiomyoma-,lipoma-, and salivary gland adenoma-cells, with the breakpoint clusterregions of the various tumor types overlapping [25, “ANNEX 1”]. This 1.7Mb region on the long arm of chromosome 12, which contains ULCR12obviously, was designated Multiple Aberration Region (MAR) to reflectthis feature. In a regional fine mapping study, MAR was recentlyassigned to 12q15.

It has thus been found that essentially all breakpoints of chromosome 12map in a 1.7 Mb region indicated herein as the “Multiple AberrationRegion” or MAR. Further research revealed that in this region a memberof the High Mobility Group gene family, the HMGI-C gene, can beidentified as a postulated multi-tumor aberrant growth gene (MAG). Thesame applies to members of the LIM family which are also found to beinvolved in chromosome aberrations. Of these the chromosome 3-derivedLipoma-Preferred Partner (LPP) gene is particularly implicated inlipomas.

LIM proteins are proteins carrying cystein-rich zinc-binding domains,so-called LIM domains. They are involved in protein-protein interactions[for a review see ref. 80]. One of the members of the protein family isthe now disclosed LPP protein mapping at chromosome 3.

According to the present invention the aberrations in the HMGI-C gene onchromosome 12 and the LPP gene on chromosome 3 have been used as a modelto reveal the more general concept of the involvement of members of theHMG and LIM gene families in both benign and malignant tumors. Todemonstrate that there exists a more general concept of gene families,which, when affected by chromosome rearrangements, lead to a particulargroup of tumor growth, the present inventors demonstrated that theHMGI(Y) gene, which is a member of the HMG family, is involved in breaksin chromosome 6.

Although both the HMG and LIM gene families are known per se, up tillthe present invention the correlation between these families and tumorinducing chromosome aberrations, like translocations, deletions,insertions and inversions, has not been anticipated. Furthermore, untilnow it was not previously demonstrated that alterations in thephysiological expression level of the members of the gene family areprobably also implicated in tumor development. According to theinvention it was demonstrated that in normal adult cells the expressionlevel of the HMGI-C gene is practically undetectable, whereas inaberrantly growing cells the expression level is significantlyincreased.

SUMMARY OF THE INVENTION

Based on these insights the present invention now provides for themembers of the gene families or derivatives thereof in isolated form andtheir use in diagnostic and therapeutic applications. Furthermore theknowledge on the location and nucleotide sequence of the genes may beused to study their rearrangements or expression and to identify apossible increase or decrease in their expression level and the effectsthereof on cell growth. Based on this information diagnostic tests ortherapeutic treatments may be designed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a long range physical map of a 6 Mb region on the long arm ofhuman chromosome 12 deduced from a YAC contig consisting of 75overlapping CEPH YAC clones and spanning the chromosome 12q breakpointspresent in a variety of benign solid tumors;

FIG. 2 is a contig of overlapping cosmids, long range restriction andSTS map spanning a segment of MAR of about 445 kb;

FIG. 3 is a schematic representation of FISH mapping data obtained fortumor cell lines with chromosome 12q13-q15 aberrations;

FIG. 4 is a sequence of 3'-RACE product comprising the junction betweenpart of the HMGI-C gene and part of the LPP gene;

FIG. 5 is a partial cDNA sequence of the LPP gene;

FIG. 6 is an amino acid sequence of the LPP gene;

FIG. 7 is a nucleotide sequence of HMGI-C (U28749); and

FIG. 8 is a photograph of a gel of PCR products obtained as described inExample 5.

FIG. 9 is a schematic representation of FISH mapping data obtained forseven pleomorphic salivary gland adenoma cell lines;

FIG. 10 is a schematic representation of a partial karyotype ofAd-295/SV40, and depiction of FISH analysis of metaphase chromosomes ofAd-295/SV40 cells;

FIG. 11 is a schematic representation of chromosome 12 breakpointmapping data obtained for primary pleomorphic salivary gland adenomas,uterine leiomyomas, and lipomas as well as cell lines derived from suchsolid tumors;

FIG. 12 is a composite physical map of the overlapping DNA inserts ofYAC clones Y4854 and Y9091;

FIG. 13 is a depiction showing mapping of cosmid clone cPK12qter to thetelomeric region of the long arm of chromosome 12, and FISH analysis ofmetaphase chromosomes of Ad-312/SV40 cells using DNA of YAC clone Y4584or Y9091 as molecular probe;

FIG. 14 is a depiction of FISH analysis of metaphase chromosomes of Ad-312/SV40 cells using DNA cosmid clone cRM69 or RM111 as molecular probe;and

FIG. 15 is a schematic representation of FISH mapping data obtained forsix pleomorphic salivary gland adenoma cell lines.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In this application the term “Multi-tumor Aberrant Growth (or MAG) gene”will be used to indicate the involvement of these types of genes invarious types of cancer. The term refers to all members of the HMG andLIM gene families involved in non-physiological proliferative growth,and in particular involved in malignant or benign tumors, includingatherosclerotic plaques. However, according to the invention it wasfurthermore found that even breaks outside the actual gene but in thevicinity thereof might result in aberrant growth. The term MAG gene istherefore also intended to include the immediate vicinity of the gene.The skilled person will understand that the “immediate vicinity” shouldbe understood to include the surroundings of the gene in which breaks orrearrangements will result in the above defined non-physiologicalproliferative growth.

The term “wildtype cell” is used to indicate the cell not harbouring anaberrant chromosome or to a cell having a physiological expression levelof the relevant gene. “Wildtype” or “normal” chromosome refers to anon-aberrant chromosome.

The present invention provides for various diagnostic and therapeuticapplications that are based on the information that may be derived fromthe genes. This information not only encompasses its nucleotide sequenceor the amino acid sequence of the gene product derived from the gene,but also involves the levels of transcription or translation of thegene.

The invention is thus two-fold. On the one hand the aberration in cellgrowth may be directly or indirectly caused by the physical breaks thatoccur in the gene or its vicinity. On the other hand the aberration incell growth may be caused by a non-physiological expression level of thegene. This non-physiological expression level may be caused by thebreak, or may be due to another stimulus that activates or deactivatesthe gene. At present the exact mechanism or origin of the aberrant cellgrowth is not yet unraveled. However, exact knowledge on this mechanismis not necessary to define methods of diagnosis or treatment.

Diagnostic methods according to the invention are thus based on the factthat an aberration in a chromosome results in a detectable alteration inthe chromosomes' appearance or biochemical behaviour. A translocation,for example will result in a first part of the chromosome (andconsequently of the MAG gene) having been substituted for another(second) part (further referred to as “first and second substitutionparts”). The first part will often appear someplace else on anotherchromosome from which the second part originates. As a consequencehybrids will be formed between the remaining parts of both (or in casesof triple translocations, even more) chromosomes and the substitutionparts provided by their translocation partners. Since it has now beenfound that the breaks occur in a MAG gene this will result in hybridgene products of that MAG gene. Markers, such as hybridising moleculeslike RNA, DNA or DNA/RNA hybrids, or antibodies will be able to detectsuch hybrids, both on the DNA level, and on the RNA or protein level.

For example, the transcript of a hybrid will still comprise the regionprovided by the remaining part of the gene/chromosome but will miss theregion provided by the substitution part that has been translocated. Inthe case of inversions, deletions and insertions the gene may be equallyafflicted.

Translocations are usually also cytogenetically detectable. The otheraberrations are more difficult to find because they are often notvisible on a cytogenetical level. The invention now providespossibilities for diagnosing all these types of chromosomal aberrations.

In translocations markers or probes based on the MAG gene for theremaining and substitution parts of a chromosome in situ detect theremaining part on the original chromosome but the substitution part onanother, the translocation partner.

In the case of inversions for example, two probes will hybridise at aspecific distance in the wildtype gene. This distance might howeverchange due to an inversion. In situ such inversion may thus bevisualized by labeling a set of suitable probes with the same ordifferent detectable markers, such as fluorescent labels. Deletions andinsertions may be detected in a similar manner.

According to the invention the above in situ applications can veryadvantageously be performed by using FISH techniques. The markers aree.g. two cosmids one of which comprises exons 1 to 3 of the MAG gene,while the other comprises exons 4 and 5. Both cosmids are labeled withdifferent fluorescent markers, e.g. blue and yellow. The normalchromosome will show a combination of both labels, thus giving a greensignal, while the translocation is visible as a blue signal on theremaining part of one chromosome (e.g. 12) while the yellow signal isfound on another chromosome comprising the substitution part. In casethe same labels are used for both probes, the intensity of the signal onthe normal chromosome will be 100%, while the signal on the aberrantchromosomes is 50%. In the case of inversions one of the signals shiftsfrom one place on the normal chromosome to another on the aberrant one.

In the above applications a reference must be included for comparison.Usually only one of the two chromosomes is afflicted. It will thus bevery convenient to use the normal chromosome as an internal reference.Furthermore it is important to select one of the markers on theremaining or unchanging part of the chromosome and the other on thesubstitution or inverted part. In the case of the MAG gene of chromosome12, breaks are usually found in the big intron between exons 3 and 4 asis shown by the present invention. Furthermore breaks were detectedbetween exons 4 and 5. Probes based on exons 1 to 3 and 4 and 5, orprobes based on either exon 4 or on exon 5 are thus very useful. Asan-alternative a combination of probes based on bothtranslocation orfusion partners may be used. For example, for the identification oflipomas one may use probes based on exons 1 to 3 of the HMGI-C gene onthe one hand and based on the LIM domains of the LPP gene on the otherhand.

Furthermore it was found that breaks might also occur outside the gene,i.e. 5′ or 3′ thereof. The choice of the probes will then of courseinclude at least one probe hybrising to a DNA sequence located 5′ or 3′of the gene.

“Probes” as used herein should be widely interpreted and include but arenot limited to linear DNA or RNA strands, Yeast Artificial Chromosomes(YACs), or circular DNA forms, such as plasmids, phages, cosmids etc.

These in situ methods may be used on metaphase and interphasechromosomes.

Besides the above described in situ methods various diagnostictechniques may be performed on a more biochemical level, for examplebased on alterations in the DNA, RNA or protein, or on changes in thephysiological expression level of the gene.

Basis for the methods that are based on alterations in the chromosome'sbiochemical behaviour is the fact that by choosing suitable probes,variations in the length or composition in the gene, transcript orprotein may be detected on a gel or blot. Variations in length arevisible because the normal gene, transcript(s) or protein(s) will appearin another place on the gel or blot then the aberrant one(s). In case ofa translocation more than the normal number of spots will appear.

Based on the above principle the invention may thus for example relateto a method of diagnosing cells having a non-physiological proliferativecapacity, comprising the steps of taking a biopsy of the cells to bediagnosed, isolating a suitable MAG gene-related macromoleculetherefrom, and analysing the macromolecule thus obtained by comparisonwith a reference molecule originating from cells not showing anon-physiological proliferative capacity, preferably from the sameindividual. The MAG gene-related macromolecule may thus be a DNA, an RNAor a protein. The MAG gene may be either a member of the HMG family orof the LIM family.

In a specific embodiment of this type of diagnostic method the inventioncomprises the steps of taking a biopsy of the cells to be diagnosed,extracting total RNA thereof, preparing a first strand cDNA of the mRNAspecies in the total RNA extract or poly-A-selected fraction(s) thereof,which cDNA comprises a suitable tail; performing a PCR using a MAG genespecific primer and a tail-specific primer in order to amplify MAG genespecific cDNA's; separating the PCR products on a gel to obtain apattern of bands; evaluating the presence of aberrant bands bycomparison to wildtype bands, preferably originating from the sameindividual.

As an alternative amplification may be performed by means of the NucleicAcid Sequence-Based Amplification (NASBA) technique [81] or variationsthereof.

In another embodiment the method comprises the steps of taking a biopsyof the cells to be diagnosed, isolating total protein therefrom,separating the total protein on a gel to obtain essentially individualbands, optionally transfering the bands to a Western blot, hybridizingthe bands thus obtained with antibodies directed against a part of theprotein encoded by the remaining part of the MAG gene and against a partof the protein encoded by the substitution part of the MAG gene;visualising the antigen-antibody reactions and establishing the presenceof aberrant bands by comparison with bands from wildtype proteins,preferably originating from the same individual.

In a further embodiment the method comprises taking a biopsy of thecells to be diagnosed; isolating total DNA therefrom; digesting the DNAwith one or more so-called “rare cutter” restriction enzymes (typically“6- or more cutters”); separating the digest thus prepared on a gel toobtain a separation pattern; optionally trangsfering the separationpattern to a Southern blot; hybridizing the separation pattern in thegel or on the blot with a set of probes under hybridizing conditions;visualising the hybridizations and establishing the presence of aberrantbands by comparison to wildtype bands, preferably originating from thesame individual.

Changes in the expression level of the gene may be detected by measuringmRNA levels or protein levels by means of a suitable probe.

Diagnostic methods based on abnormal expression levels of the gene maycomprise the steps of taking a sample of the cells to be diagnosed;isolating mRNA therefrom; and establishing the presence and/or the(relative) quantity of mRNA transcribed from the MAG gene of interest incomparison to a control. Establishing the presence or (relative)quantity of the mRNA may be achieved by amplifying at least part of themRNA of the MAG gene by means of RT-PCR or similar amplificationtechniques. In an alternative embodiment the expression level may beestablished by determination of the presence or the amount of the geneproduct (e.g. protein) by means of for example monoclonal antibodies.

The diagnostic methods of the invention may be used for diseases whereincells having a non-physiological proliferative capacity are selectedfrom the group consisting of benign tumors, such as the mesenchymaltumors hamartomas (e.g. breast and lung), adipose tissue tumors (e.g.lipomas), pleomorphic salivary gland adenomas, uterine leiomyomas,angiomyxomas, fibroadenomas of the breast, polyps of the endometrium,atherosclerotic plaques, and other benign tumors as well as variousmalignant tumors, including but not limited to sarcomas (e.g.rhabdomyosarcoma, osteosarcoma) and carcinomas (e.g. of breast, lung,skin, thyroid). The invention is not limited to the diagnosis andtreatment of so-called benign and malignant solid tumors, but theprinciples thereof have been found to also apply to haematologicalmalignancies like leukemias and lymphomas.

Recent publications indicate that atherosclerotic plaques also involveabnormal proliferation [26] of mainly smooth muscle cells and it waspostulated that atherosclerotic plaques constitute benign tumors [27].Therefore, this type of disorder is also to be understood as a possibleindication for the use of the MAG gene family, in particular indiagnostic and therapeutic applications.

As already indicated above it has been found that in certain malignanttumors the expression level of the HMG genes is increased [28]. Untilnow the relevance of this observation was not understood. Another aspectof the invention thus relates to the implementation of theidentification of the MAG genes in therapy. The invention for exampleprovides anti-sense molecules or expression inhibitors of the MAG genefor use in the treatment of diseases involving cells having anon-physiological proliferative capacity by modulating the expression ofthe gene. A non-physiological high expression may thus be normalised bymeans of antisense RNA that is either administered to the cell orexpressed thereby and binds to the mRNA, or antibodies directed to thegene product, which in turn may result in a normalization of the cellgrowth. The examples will demonstrate that expression of antisense RNAin leukemic cells results in a re-differentiation of the cells back tonormal.

The invention thus provides derivatives of the MAG gene and/or itsimmediate environment for use in diagnosis and the preparation oftherapeutical compositions, wherein the derivatives are selected fromthe group consisting of sense and anti-sense cDNA or fragments thereof,transcripts of the gene or fragments thereof, antisense RNA, triplehelix inducing molecule or other types of “transcription clamps”,fragments of the gene or its complementary strand, proteins encoded bythe gene or fragments thereof, protein nucleic acids (PNA), antibodiesdirected to the gene, the cDNA, the transcript, the protein or thefragments thereof, as well as antibody fragments. Besides the use ofdirect derivatives of the genes and their surroundings (flankingsequences) in diagnosis and therapy, other molecules, like expressioninhibitors or expression enhancers, may be used for therapeutictreatment according to the invention. An example of this type ofmolecule are ribozymes that destroy RNA molecules.

Besides the above described therapeutic and diagnostic methods theprinciples of the invention may also be used for producing a transgenicanimal model for testing pharmaceuticals for treatment of MAG generelated malignant or benign tumors and atherosclerotic plaques. One ofthe examples describes the production of such an animal model.

It is to be understood that the principles of the present invention aredescribed herein for illustration purposes only with reference to theHMGI-C gene mapping at chromosome 12 and the HMGI(Y) gene mapping atchromosome 6 and the LPP gene on chromosome 3. Based on the informationprovided in this application the skilled person will be able to isolateand sequence corresponding genes of the gene family and apply theprinciples of this invention by using the gene and its sequence withoutdeparting from the scope of the general concept of this invention.

The present invention will thus be further elucidated by the followingexamples which are in no way intended to limit the scope thereof.

EXAMPLES Example 1

1. Introduction

This example describes the isolation and analysis of 75 overlapping YACclones and the establishment of a YAC contig (set of overlappingclones), which spans about 6 Mb of genomic DNA around locus D12S8 andincludes MAR. The orientation of the YAC contig on the long arm ofchromosome 12 was determined by double-color FISH analysis. On the basisof STS-content mapping and restriction enzyme analysis, a long rangephysical map of this 6 Mb DNA region was established. The contigrepresents a useful resource for cDNA capture aimed at identifying geneslocated in 12q15, including the one directly affected by the variouschromosome 12 aberrations.

2. Materials and Methods

2.1. Cell Lines

Cell lines PK89-12 and LIS-3/SV40/A9-B4 were used for ChromosomeAssignment using Somatic cell Hybrids (CASH) experiments. PK89-12, whichcontains chromosome 12 as the sole human chromosome in a hamster geneticbackground, has been described before [29]. PK89-12 cells were grown inDME-F12 medium supplemented with 10% fetal bovine serum, 200 IU/mlpenicillin, and 200 μg/ml streptomycin. Somatic cell hybridLIS-3/SV40/A9-B4 was obtained upon fusion of myxoid liposarcoma cellline LIS-3/SV40, which carries a t(12;16)(q13;p11.2), and mouse A9 cellsand was previously shown to contain der(16), but neither der(12) nor thenormal chromosome 12 [30]. LIS-3/SV40/A9-B4 cells were grown inselective AOA-medium (AOA-medium which consisted of DME-F12 mediumsupplemented with 10% fetal bovine serum, 0.05 mM adenine, 0.05 mMouabain, and 0.01 mM azaserine). Both cell lines were frequently assayedby standard cytogenetic techniques.

2.2. Nucleotide Sequence Analysis and Oligonucleotides.

Nucleotide sequences were determined according to the dideoxy chaintermination method using a T7 polymerase sequencing kit (Pharmacia/LKB)or a dsDNA Cycle Sequencing System (GIBCO/BRL). DNA fragments weresubcloned in pGEM-3Zf(+) and sequenced using FITC-labelled standard SP6or T7 primers, or specific primers synthesized based upon newly obtainedsequences. Sequencing results were obtained using an Automated LaserFluorescent (A.L.F.) DNA sequencer (Pharmacia Biotech) and standard 30cm, 6% Hydrolink^(R), Long Range™ gels (AT Biochem). The nucleotidesequences were analyzed using the sequence analysis software Genepro(Riverside Scientific), PC/Gene (IntelliGenetics), the IntelliGeneticsSuite software package (IntelliGenetics, Inc.), and oligo [31]. Alloligonucleotides were purchased from Pharmacia Biotech.

2.3. Chromosome Preparations and Fluorescence in situ Hybridization(FISH)

FISH analysis of YAC clones was performed to establish their chromosomalpositions and to identify chimeric clones. FISH analysis(of cosmidclones corresponding to STSs of YAC insert ends were performed toestablish their chromosomal positions. Cosmids were isolated from humangenomic library CMLW-25383 [32] or the arrayed chromosome 12-specificlibrary constructed at Lawrence Livermore.National Laboratory (LL12NC01,ref. 33) according to standard procedures [34]. Routine FISH analysiswas performed essentially as described before [30, 35]. DNA was labelledwith biotin-11-dUTP (Boehringer) using the protocol of Kievits et al.[36]. Antifade medium, consisting of DABCO (2 g/100 ml, Sigma), 0.1 MTris-HCL pH 8, 0.02% Thimerosal, and glycerol (90%), and containingpropidium iodide (0.5 μg/ml, Sigma) as a counterstain, was added 15 minbefore specimens were analyzed on a Zeiss Axiophot fluorescencemicroscope using a double band-pass filter for FITC/Texas red (OmegaOptical, Inc.). Results were recorded on Scotch (3M) 640 ASA film.

For the double colour FISH experiments, LLNL12NCO1-96C11 was labelledwith digoxygenin-11-dUTP (Boehringer) and cosmids LLNL12NCO1-1F6 and-193F10, with biotin-11-dUTP. Equal amounts of each probe were combinedand this mixture was used for hybridization. After hybridization, slideswere incubated for 20 min with Avidin-FITC and then washed as describedby Kievits et al. [36]. Subsequent series of incubations in TNB buffer(0.1 M Tris-HCl pH 7.5, 0.15 M NaCl, 0.5% Boehringer blocking agent(Boehringer)) and washing steps were performed in TNT buffer (0.1 MTris-HCl pH 7.5, 0.15 M NaCl, 0.05% Tween-20); all incubations wereperformed at 37° C. for 30 min. During the second incubation,Goat-α-Avidin-biotin (Vector) and Mouse-α-digoxygenin (Sigma) wereapplied simultaneously. During the third incubation, Avidin-FITC andRabbit-α-Mouse-TRITC (Sigma) were applied. During the last incubation,Goat-α-Rabbit-TRITC (Sigma) was applied. After a last wash in TNTbuffer, samples were washed twice in 1×PBS and then dehydrated throughan ethanol series (70%, 90%, 100%). Antifade medium containing 75 ng/μlDAPI (Serva) as counterstain was used. Specimens were analyzed on aZeiss Axiophot fluorescence microscope as described above.

2.4. Screening of YAC Libraries.

YAC clones were isolated from CEPH human genomic YAC libraries mark 1and 3 [37, 38] made available to us by the Centre d'Étude duPolyphormisme Humain (CEPH). Screening was carried out as previouslydescribed [39]. Contaminating Candida parapsylosis, which was sometimesencountered, was eradicated by adding terbinafin (kindly supplied by Dr.Dieter Römer, Sandoz Pharma LTD, Basle, Switzerland) to the growthmedium (final concentration: 25 μg/ml). The isolated YAC clones werecharacterized by STS-content mapping, contour-clamped homogeneouselectric field (CHEF) gel electrophoresis [40], restriction mapping, andhybridization- and FISH analysis.

2.5. PCR Reactions

PCR amplification was carried out using a Pharmacia/LKB Gene ATAQController (Pharmacia/LKB) in final volumes of 100 μl containing 10 mMTris-HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl₂, 0.01% gelatine, 2 MM dNTP's,20 pmole of each amplimer, 2.5 units of Amplitaq (Perkin-Elmer Cetus),and 100 ng (for superpools) or 20 ng (for pools) of DNA. After initialdenaturation for 5 min at 94° C., 35 amplification cycles were performedeach consisting of denaturation for 1 min at 94° C., annealing for 1 minat the appropriate temperature (see Table I) and extension for 1 min at72° C. The PCR reaction was completed by a final extension at 72° C. for5 min. Results were evaluated by analysis of 10 μl of the reactionproduct on polyacrylamide minigels.

2.6. Pulsed-field Gel Electrophoresis and Southern Blot Analysis

Pulsed-field gel electrophoresis and Southern blot analysis wereperformed exactly as described by Schoenmakers et al. [39]. Agaroseplugs containing high-molecular weight YAC DNA (equivalent to about1×10⁸ yeast cells) were twice equilibrated in approximately 25 ml TEbuffer (pH 8.0) for min at 50° C. followed by two similar rounds ofequilibration at room temperature. Plugs were subsequently transferredto round-bottom 2 ml eppendorf tubes and equilibrated two times for 30min in 500 μl of the appropriate 1×restriction-buffer at the appropriaterestriction temperature. Thereafter, DNA was digested in the plugsaccording to the suppliers (Boehringer) instructions for 4 h using 30units of restriction endonuclease per digestion reaction. Afterdigestion, plugs along with appropriate molecular weight markers wereloaded onto a 1% agarose/0.25×TBE gel, sealed with LMP-agarose and sizefractionated on a CHEF apparatus (Biorad) for 18 h at 6.0 V/cm using apulse angle of 120 degrees and constant pulse times varying from 10 sec(separation up to 300 kbp) to 20 sec (separation up to 500 kbp). In thecase of large restriction fragments, additional runs were performed,aiming at the separation of fragments with sizes above 500 kbp.Electrophoresis was performed at 14° C. in 0.25×TBE. As molecular weightmarkers, lambda ladders (Promega) and home-made plugs containing lambdaDNA cut with restriction endonuclease HindIII were used. Afterelectrophoresis, gels were stained with ethidium bromide, photographed,and UV irradiated using a stratalinker (Stratagene) set at 120 mJ. DNAwas subsequently blotted onto Hybond N⁺ membranes (Amersham) for 4-16 husing 0.4 N NaOH as transfer buffer. After blotting, the membranes weredried for 15 min at 80° C., briefly neutralised in 2×SSPE, andprehybridised for at least 3 h at 42° C. in 50 ml of a solutionconsisting of 50% formamide, 5×SSPE, 5×Denhardts, 0.1% SDS and 200 μg/mlheparin. Filters were subsequently hybridised for 16 h at 42° C. in 10ml of a solution consisting of 50% formamide, 5×SSPE, 1×Denhardts, 0.1%SDS, 100 μg/ml heparin, 0.5% dextran sulphate and 2-3×10⁶ cpm/ml oflabelled probe. Thereafter, membranes were first washed two times for 5min in 2×SSPE/0.1% SDS at room temperature, then for 30 min in2×SSPE/0.1% SDS at 42° C. and, finally, in 0.1×SSPE/0.1% SDS for 20 minat 65° C. Kodak XAR-5 films were exposed at −80° C. for 3-16 h,depending on probe performance. Intensifying screens (Kyokko special500) were used.

2.7. Generation of STSs from YAC Insert Ends

STSs from YAC insert ends were obtained using a vectorette-PCR procedurein combination with direct DNA sequencing analysis, essentially asdescribed by Geurts et al. [41]. Primer sets were developed and testedon human genomic DNA, basically according to procedures described above.STSs will be referred to throughout this application by theirabbreviated names (for instance: RM1 instead of STS 12-RM1) for reasonsof convenience.

3. Results

3.1. Assembly of a YAC Contig Around Locus D12S8

In previous studies [39], a 800 kb YAC contig around D12S8 wasdescribed. This contig consisted of the following three partiallyoverlapping, non-chimeric CEPH YAC clones: 258F11, 320F6, and 234G11.This contig was used as starting point for a chromosome walking projectto define the DNA region on the long arm of chromosome 12 thatencompasses the breakpoints of a variety of benign solid tumors, whichare all located proximal to D12S8 and distal to CHOP. Initially,chromosome walking was performed bidirectionally until the size of thecontig allowed reliable determination of the orientation of it. In thebidirectional and subsequent unidirectional chromosome walking steps,the following general procedure was used. First, rescuing and sequencingthe ends of YAC clones resulted in DNA markers characterizing the leftand right sides of these (Table I). Based on sequence data of the endsof forty YAC inserts, primer sets were developed for specificamplification of DNA; establishing STSs (SEQ ID Nos:17-102) (Table II).Their localization to 12q13-qter was determined by CASH as well as FISHafter corresponding cosmid clones were isolated. It should be noted thatisolated YAC clones were often evaluated by FISH analysis too, thus notonly revealing the chromosomal origin of their inserts but also, for anumber of cases, establishing and defining their chimeric nature.Moreover, it should be emphasized that data obtained by restrictionendonuclease analysis of overlapping YAC clones were also taken intoaccount in the YAC clone evaluation and subsequent alignment. With thesequentially selected and evaluated primer sets, screening of the YACand cosmid libraries was performed to isolate the building blocks forcontig-assembly. Therefore, contig-assembly was performed using dataderived from FISH- and STS-content mapping as well as restrictionendonuclease analysis. Using this approach, we established a YAC contigconsisting of 75 overlapping YAC clones, covering approximately 6 Mb ofDNA (FIG. 1). This contig appeared to encompass the chromosome 12breakpoints of all tumor-derived cell lines studied [39].Characteristics of the YACs that were used to build this contig aregiven in Table I.

3.2. Establishment of the Chromosomal Orientation of the YAC Contig

To allow unidirectional chromosome walking towards the centromere ofchromosome 12, the orientation of the DNA region flanked by STSs RM14and RM26 (approximate size: 1450 kb) was determined by double-colorinterphase FISH analysis. Cosmid clones corresponding to these STSs(i.e. LL12NC01-1F6 (RM14) and LL12NC01-96C11 (RM26)) were differentiallylabelled to show green or red signals, respectively. As a referencelocus, cosmid LL12NC01-193F10 was labelled to show green signals upondetection. LL12NC01-193F10 had previously been mapped distal to thebreakpoint of LIS-3/SV40 (i.e. CHOP) and proximal to the chromosome 12qbreakpoints in lipoma cell line Li-14/SV40 and uterine leiomyoma cellline LM-30.1/SV40. LL12NC01-1F6 and LL12NC01-96C11 were found to bemapping distal to the 12q breakpoints in lipoma cell line Li-14/SV40 anduterine leiomyoma cell line LM-30.1/SV40. Therefore, LL12NC01-193F10 wasconcluded to be mapping proximal to both RM14 and RM26 (unpublishedresults). Of 150 informative interphases scored, 18% showed asignal-order of red-green-green whereas 72% showed a signal order ofgreen-red-green. Based upon these observations, we concluded that RM26mapped proximal to RM14, and therefore we continued to extend the YACcontig from the RM26 (i.e. proximal) side of our contig only. Only thecosmids containing RM14 and RM26 were ordered by double-color interphasemapping; the order of all others was deduced from data of the YACcontig. Finally, it should be noted that the chromosomal orientation ofthe contig as proposed on the basis of results of the double-colorinterphase FISH studies was independently confirmed after the YAC contighad been extended across the chromosome 12 breakpoints as present in avariety of tumor cell lines. This confirmatory information was obtainedin extensive FISH studies in which the positions of YAC and cosmidclones were determined relative to the chromosome 12q13-q15 breakpointsof primary lipomas, uterine leiomyomas, pleomorphic salivary glandadenomas, and pulmonary chondroid hamartomas or derivative cell lines[24, 42, 25, 43].

3.3. Construction of a Rare-Cutter Physical Map from the 6 Mb YAC ContigAround D12S8

Southern blots of total yeast plus YAC DNA, digested to completion withrare-cutter enzymes (see Materials and Methods) and separated on CHEFgels, were hybridized sequentially with i) the STS used for the initialscreening of the YAC in question, ii) pYAC4 right arm sequences, iii)pYAC4 left arm sequences, and iv) a human ALU-repeat probe (BLUR-8). Thelong-range restriction map that was obtained in this way, was completedby probing with PCR-isolated STSs/YAC end probes. Occasionallydouble-digests were performed.

Restriction maps of individual YAC clones were aligned and a consensusrestriction map was established. It is important to note here that theentire consensus rare-cutter map was supported by at least twoindependent clones showing a full internal consistency.

3.4. Physical Mapping of CA Repeats and Monomorphic STSs/ESTs

Based upon integrated mapping data as emerged from the SecondInternational Workshop on Human Chromosome 12 [44], a number ofpublished markers was expected to be mapping within the YAC contigpresented here. To allow full integration of our mapping data with thoseobtained by others, a number of markers were STS content-mapped on ourcontig, and the ones found positive were subsequently sublocalized by(primer-)hybridization on YAC Southern blots. Among the markers thatwere found to reside within the contig presented here were CA repeatsD12S313 (AFM207f2) and D12S335 (AFM273vg9) [45], D12S375 (CHLCGATA3F02), and D12S56 [46]. Furthermore, the interferon gamma gene(IFNG) [47], the ras-related protein gene Rap1B [48], and expressedsequence tag EST01096 [49] were mapped using primer sets which wedeveloped based on publicly available sequence data (see Table II).Markers which were tested and found negative included D12S80 (AFM102d6),D12S92 (AFM203va7), D12S329 (AFM249h9) and D12S344 (AFM296d9).

4. Discussion

In the present example the establishment of a YAC contig and rare-cutterphysical map covering approximately 6 Mb on 12q15, a region on the longarm of human chromosome 12 containing MAR in which a number of recurrentchromosomal aberrations of benign solid tumors are known to be mappingwas illustrated.

The extent of overlap between individual YACs has been carefullydetermined, placing the total length of the contig at approximately 6 Mb(FIG. 1). It should be noted that our sizing-data for some of the YACclones differ slightly from the sizes determined by CEPH [50]. It is ourbelief that this is most probably due to differences in the parametersfor running the pulsed-field gels in the different laboratories.

Using restriction mapping and STS-content analysis, a consensus longrange physical map (FIG. 1) was constructed. The entire composite map issupported by at least two-fold coverage. In total over 30 Mb of YAC DNAwas characterized by restriction and STS content analysis, correspondingto an average contig coverage of about 5 times. Although the “inborn”limited resolution associated with the technique of pulsed-fieldelectrophoresis does not allow very precise size estimations, comparisonto restriction mapping data obtained from a 500 kb cosmid contigcontained within the YAC contig presented here showed a remarkable goodcorrelation. Extrapolating from the cosmid data, we estimate theaccuracy of the rare-cutter physical map presented here at about 10 kb.

The results of our physical mapping studies allowed integration of threegene-specific as well as five anonymous markers isolated by others(indicated in between arrows in FIG. 1). The anonymous markers includeone monomorphic and four polymorphic markers. Five previously publishedYAC-end-derived single copy STSs (RM1, RM4, RM5, RM7, and RM21) as wellas four published CA repeats (D12S56, D12S313, D12S335, and D12S375) andthree published gene-associated STSs/ESTs (RAPLB, EST01096, and IFNG)have been placed on the same physical map and this will facilitate(linkage-) mapping and identification of a number of traits/diseasegenes that map in the region. Furthermore, we were able to place ontothe same physical map, seventy two YAC-end-derived (Table I) and eightcosmid-end- or inter-ALU-derived DNA markers (CH9, RM1, RM110, RM111,RM130, RM131, RM132, and RM133), which were developed during the processof chromosome walking. The PYTHIA automatic mail server atPYTHIA@anl.gov was used to screen the derived sequences of these DNAmarkers for the presence of repeats. For forty three of these seventytwo DNA markers (listed in Table II), primer sets were developed and thecorresponding STSs were determined to be single copy by PCR as well asSouthern blot analysis of human genomic DNA. The twenty nine remainingDNA markers (depicted in the yellow boxes) represent YAC-end-derivedsequences for which we did not develop primer sets. These YAC-endsequences are assumed to be mapping to chromosome 12 on the basis ofrestriction mapping. The final picture reveals an overall marker densityin this region of approximately one within every 70 kb.

The analysis of the contig presented here revealed many CpG-richregions, potentially HTF islands, which are known to be frequentlyassociated with housekeeping genes. These CpG islands are most probablylocated at the 5′ ends of as yet unidentified genes: it has been shownthat in 90% of cases in which three or more rare-cutter restrictionsites coincide in YAC DNA there is an associated gene [51]. This islikely to be an underestimate of the number of genes yet to beidentified in this region because 60% of tissue-specific genes are notassociated with CpG islands [52] and also because it is possible for twogenes to be transcribed in different orientations from a single island[53].

While several of the YAC clones that were isolated from the CEPH YAClibrary mark 1 were found to be chimeric, overlapping YAC clones thatappeared to be non-chimeric based on FISH, restriction mapping and STScontent analysis could be obtained in each screening, which is inagreement with the reported complexity of the library. The degree ofchimerism for the CEPH YAC library mark 1 was determined at 18% (12 outof 68) for the region under investigation here. The small number of YACsfrom the CEPH YAC library mark 3 (only 7 MEGA YACs were included in thisstudy) did not allow a reliable estimation of the percentage of chimericclones present in this library. The average size of YACs derived fromthe mark 1 library was calculated to be 381 kb ; non-chimeric YACs(n=58) had an average size of 366 kb while chimeric YACs (n=12) werefound to have a considerable larger average size; i.e. 454 kb.

In summary, we present a 6 Mb YAC contig corresponding to a humanchromosomal region which is frequently rearranged in a variety of benignsolid tumors. The contig links over 84 loci, including 3 gene-associatedSTSs. Moreover, by restriction mapping we have identified at least 12CpG islands which might be indicative for genes residing there. Finally,four CA repeats have been sublocalized within the contig. Theintegration of the genetic, physical, and transcriptional maps of theregion provides a basic framework for further studies of this region ofchromosome 12. Initial studies are likely to focus on MAR and ULCR12, asthese regions contain the breakpoint cluster regions of at least threedistinct types of solid tumors. The various YAC clones we describe hereare valuable resources for such studies. They should facilitate thesearch for genes residing in this area and the identification of thosedirectly affected by the chromosome 12q aberrations of the variousbenign solid tumors.

Example 2

1. Introduction

It was found that the 1.7 Mb Multiple Aberration Region on humanchromosome 12q15 harbors recurrent chromosome 12 breakpoints frequentlyfound in different benign solid tumor types. In this example theidentification of an HMG gene within MAR that appears to be ofpathogenetical relevance is described. Using a positional cloningapproach, the High Mobility Group protein gene HMGI-C was identifiedwithin a 175 kb segment of MAR and its genomic organizationcharacterized. By FISH, within this gene the majority of the breakpointsof seven different benign solid tumor types were pinpointed. By Southernblot and 3′-RACE analysis, consistent rearrangements in HMGI-C and/orexpression of altered HMGI-C transcripts were demonstrated. Theseresults indicate a link between a member of the HMG gene family andbenign solid tumor development.

2. Materials and Methods

2.1. Cell Culture and Primary Tumor Specimens.

Tumor cell lines listed in FIG. 3 were established by a transfectionprocedure [54] and have been described before in [39, 24] and in anarticle of Van de Ven et al., Genes Chromosom. Cancer 12, 296-303 (1995)enclosed with this application as ANNEX 1. Cells were grown in TC199medium supplemented with 20% fetal bovine serum and were assayed bystandard cytogenetic techniques at regular intervals. The humanhepatocellular carcinoma cell lines Hep 3B and Hep G2 were obtained fromthe ATCC (accession numbers ATCC HB 8064 and ATCC HB 8065) and culturedin DMEM/F12 supplemented with 4% Ultroser (Gibco/BRL). Primary solidtumors were obtained from various University Clinics.

2.2. YAC and Cosmid Clones

YAC clones were isolated from the CEPH mark 1 [57] and mark 3 [58] YAClibraries using a combination of PCR-based screening [59] and colonyhybridization analysis. Cosmid clones were isolated from an arrayedhuman chromosome 12-specific cosmid library (LL12NC01) [60] obtainedfrom Lawrence Livermore National Laboratory (courtesy P. de Jong).LL12NC01-derived cosmid clones are indicated by their microtiter plateaddresses; i.e. for instance 27E12.

Cosmid DNA was extracted using standard techniques involvingpurification over Qiagen tips (Diagen). Agarose plugs containinghigh-molecular weight yeast+YAC DNA (equivalent to 1×10⁹ cells ml⁻¹)were prepared as described before [61]. Plugs were thoroughly dialysedagainst four changes of 25 ml T₁₀E₁ (pH 8.0) followed by two changes of0.5 ml 1×restriction buffer before they were subjected to eitherpulsed-field restriction enzyme mapping or YAC-end rescue. YAC-endrescue was performed using a vectorette-PCR procedure in combinationwith direct solid phase DNA sequencing, as described before in reference61. Inter-Alu PCR products were isolated using publishedoligonucleotides TC65 or 517 [62] to which SalI-tails were added tofacilitate cloning. After sequence analysis, primer pairs were developedusing the OLIGO computer algorithm [61].

2.3. DNA Labelling

DNA from YACs, cosmids, PCR products and oligonucleotides was labelledusing a variety of techniques. For FISH, cosmid clones or inter-Alu PCRproducts of YACs were biotinylated with biotin-11-dUTP (Boehringer) bynick translation. For filter hybridizations, probes were radio-labelledwith α-³²P-dCTP using random hexamers [62]. In case of PCR-productssmaller than 200 bp in size, a similar protocol was applied, butspecific oligonucleotides were used to prime labelling reactions.Oligonucleotides were labelled using γ-³¹P-ATP.

2.4. Nucleotide Sequence Analysis and PCR Amplification

Nucleotide sequences were determined as described in Example 1.Sequencing results were analyzed using an A.L.F. DNA sequencer™(Pharmacia Biotech) on standard 30 cm, 6% Hydrolink^(R), Long Range™gels (AT Biochem). PCR amplifications were carried out essentially asdescribed before [39].

2.5. Rapid Amplification of cDNA Ends (RACE)

Rapid amplification of 3′ cDNA-ends (3′-RACE) was performed using aslight.modification of part of the GIBCO/BRL 33-ET protocol. For firststrand cDNA synthesis, adapter primer (AP2) AAG GAT CCG TCG ACA TC(T)₁₇(SEQ ID NO:1) was used. For both initial and secondary rounds of PCR,the universal amplification primer (UAP2) CUA CUA CUA CUA AAG GAT CCGTCG ACA TCA (SEQ ID NO:2) was used as “reversed primer”. In the firstPCR round the following specific “forward primers” were used: i) 5′-CTTCAG CCC AGG GAG AAC-3′ (exon 1)(SEQ ID NO:3), ii) 5′-CAA GAG GCA GAC CTAGGA-3′ (exon 3)(SEQ ID NO:4), or iii) 5′ -AAC AAT GCA ACT TTT AAT TACTG-3′ (3′-UTR)(SEQ ID NO:5), In the second PCR round the followingspecific forward primers (nested primers as compared to those used inthe first round) were used: i) 5′-CAU CAU CAU CAU CGC CTC AGA AGA GAGGAC-3′ (exon 1)(SEQ ID NO:6), ii) 5′-CAU CAU CAU CAU GTT CAG AAG AAG CCTGCT-3′ (exon 4)(SEQ ID NO:7), or iii) 51-CAU CAU CAU CAU TTG ATC TGA TAAGCA AGA GTG GG-3′ (3′-UTR)(SEQ ID NO:8). CUA/CAU-tailing of the nested,specific primers allowed the use of the directional CloneAmp cloningsystem (GIBCO/BRL).

3. Results

3.1. Development of Cosmid Contig and STS Map of MAR Segment

During the course of a positional cloning effort focusing on the longarm of human chromosome 12, we constructed a yeast artificial chromosome(YAC) contig spanning about 6 Mb and consisting of 75 overlapping YACS.For a description thereof reference is made to Example 1. This contigencompasses MAR (see also FIG. 2), in which most of the chromosome12q13-q15 breakpoints as present in a variety of primary benign solidtumors (34 tumors of eight different types tested sofar; Table 5) andtumor cell lines (26 tested sofar, derived from lipoma, uterineleiomyoma, and pleomorphic salivary gland adenoma; FIG. 3) appear tocluster. We have developed both a long-range STS and rare cutterphysical map of MAR and found, by FISH analysis, most of the breakpointsmapping within the 445 kb subregion of MAR located between STSs RM33 andRM98 (see FIG. 2 and 3). FISH experiments, including extensive qualitycontrol, were performed according to routine procedures as describedbefore [25, 39, 24, 42, 36] To further refine the distribution ofbreakpoints within this 445 kb MAR segment, a cosmid contig consistingof 54 overlapping cosmid clones has been developed and a dense STS map(FIG. 2) established.

The cosmid contig was double-checked by comparison to the rare cutterphysical map and by STS content mapping.

3.2. Clustering of the Chromosome 12q Breakpoints Within a 175 kb DNASegment of MAR

The chromosome 12q breakpoints in the various tumor cell lines studiedwas pinpointed within the cosmid contig by FISH (FIG. 3). As part of ourquality control FISH experiments [25, 39, 24, 423, selected cosmids werefirst tested on metaphase spreads derived from normal lymphocytes. FISHresults indicated that the majority (atleast 18 out of the 26 cases) ofthe chromosome 12 breakpoints in these tumor cell lines were found to beclustering within the 175 kb DNA interval between RM99 and RM133,indicating this interval to constitute the main breakpoint clusterregion. FISH results obtained with Li-501/SV40 indicated that part ofMAR was translocated to an apparently normal chromosome 3; a chromosomeaberration overseen by applied cytogenetics. Of interest to note,finally, is the fact that the breakpoints of uterine leiomyoma celllines LM-5.1/SV40, LM-65/SV40, and LM-608/SV40 were found to be mappingwithin the same cosmid clone; i.e. cosmid 27E12.

We also performed FISH experiments on eight different types of primarybenign solid tumors with chromosome 12q13-q15 aberrations (Table 4). Amixture of cosmid clones 27E12 and 142H1 was used as molecular probe. Insummary, the results of the FISH studies of primary tumors wereconsistent with those obtained for the tumor cell lines. The observationthat breakpoints of each of the seven different tumor types tested werefound within the same 175 kb DNA interval of MAR suggested that thisinterval is critically relevant to the development of these tumors and,therefore, might harbor the putative MAG locus or critical part(s) ofit.

3.3. Identification of Candidate Genes Mapping Within MAR

In an attempt to identify candidate genes mapping within the 175 kbsubregion of MAR between STSs RM99 and RM133, we used 3′-terminal exontrapping and genomic sequence sampling (GSS) [63]. Using the GSSapproach, we obtained DNA sequence data of the termini of a 4.9 kb BamHIsubfragment of cosmid 27E12, which was shown by FISH analysis to besplit by the chromosome 12 aberrations in three of the uterine leiomyomacell lines tested. A BLAST [64] search revealed that part of thesesequences displayed sequence identity with a publicly available partialcDNA sequence (EMBL accession #. Z31595) of the high mobility group(HMG) protein gene HMGI-C [65], which is a member of the HMG gene family[66]. In light of these observations, HMGI-C was considered a candidateMAG gene and studied in further detail.

3.4. Genomic Organization of HMGI-C and Rearrangements in Benign SolidTumors

Since only 1200 nucleotides of the HMGI-C transcript (reported sizeapproximately 4 kb [65, 67]) were publicly available, we firstdetermined most of the remaining nucleotide sequences of the HMGI-Ctranscript (Gensank, #U28749). This allowed us to subsequently establishthe genomic organization of the gene. Of interest to note about thesequence data is that a CT-repeat is present in the 5′-UTR of HMGI-C anda GGGGT-pentanucleotide repeat in the 3′-UTR, which might be ofregulatory relevance. Comparison of transcribed to genomic DNA sequences(GenBank, #U28750, U28751, U28752, U28753, and U28754) of the generevealed that HMGI-C contains at least 5 exons (FIG. 2). Transcriptionalorientation of the gene is directed towards the telomere of the long armof the chromosome. Each of the first three exons encode a putative DNAbinding domain (DBD), and exon 5 encodes an acidic domain, which isseparated from the three DBDs by a spacer domain encoded by exon 4. Thethree DBD-encoding exons are positioned relatively close together andare separated by a large intron of about 140 kb from the two otherexons, which in turn are separated about 11 kb from each other. Ofparticular interest to emphasize here is that the five exons aredispersed over a genomic region of at least 160 kb, thus almost coveringthe entire 175 kb main MAM breakpoint cluster region described above.Results of molecular cytogenetic studies, using a mixture of cosmids142H1 (containing exons 1-3) and 27E12 (containing exons 4 and 5) asmolecular probe, clearly demonstrate that the-HMGI-C gene is directlyaffected by the observed chromosome 12 aberrations in the majority ofthe tumors and tumor cell lines that were evaluated (FIG. 3; Table 4).These cytogenetic observations were independently confirmed by Southernblot analysis in the case of LM-608/SV40 (results not shown)LM-30.1/SV40 [24], and Ad-312/SV40; probes used included CH76, RM118-A,and EM26. The failure to detect the breakpoints of LM-65/SV40,LM-609/SV40, Ad-211/SV40, Ad-263/SV40, Ad-302/SV40, Li-14/SV40, andLi-538/SV40 with any of these three probes was also consistent with theFISH data establishing the relative positions of the breakpoints in MAR(cf. FIG. 3). These results made HMGI-C a prime candidate to be thepostulated MAG gene.

3.5. Expression of Aberrant HMGI-C Transcripts in Benign Solid TumorCells.

In the context of follow-up studies, it was of interest to test forpossible aberrant HMGI-C expression. Initial Northern blot studiesrevealed that transcripts of MGI-C could not be detected in a variety ofnormal tissues (brain, heart, lung, liver, kidney, pancreas, placenta,skeletal muscle) tested as well as in a number of the tumor ell lineslisted in FIG. 3 (data not shown). It is known hat HMGI-C mRNA levels innormal differentiated tissues are very much lower than in malignanttissues [65, 67]. As a control in our Northern studies, we includedhepatoma cell line Hep 3B, which is known to express relatively highlevels of HMGI-C. We readily detected two major HMGI-C transcripts,approximately 3.6 and 3.2 kb in size; with the differences in molecularweight most likely due to differences in their 5′-noncoding regions. Inan alternative and more sensitive approach to detect HMGI-C or3′-aberrant HMGI-C transcripts, we have performed 3′-RACE experiments.In control experiments using a number of tissues with varying HMGI-Ctranscription levels (high levels in Hep 3B hepatoma cells, intermediatein Hep G2 hepatoma cells, and low in myometrium, normal fat tissue, andpseudomyxoma), we obtained 3′-RACE clones which, upon molecular cloningand nucleotide sequence analysis, appeared to represent perfect partialcDNA copies of 3′-HMGI-C mRNA sequences; no matter which of the threeselected primer sets was used (see Methodology). RACE products mostlikely corresponding to cryptic or aberrantly spliced HMGI-C transcriptswere occasionally observed; their ectopic sequences were mapped back toHMGI-C intron 3 or 4.

In similar 3′-RACE analysis of ten different primary tumors or tumorcell lines derived from lipoma, uterine leiomyoma, and pleomorphicsalivary gland adenoma, we detected both constant and unique PCRproducts. The constant PCR products appeared to represent, in mostcases, perfect partial cDNA copies of 3′-HMGI-C mRNA sequences. Theymost likely originated from a presumably unaffected HMGI-C allele andmight be considered as internal controls. The unique PCR products of theten tumor cell samples presented here appeared to contain ectopicsequences fused to HMGI-C sequences. In most cases, the ectopicsequences were found to be derived from the established translocationpartners, thus providing independent evidence for translocation-inducedrearrangements of the HMGI-C gene. Information concerning nucleotidesequences (SEQ ID Nos: 103-159), diversion points, and chromosomalorigins of the ectopic sequences of these RACE products is summarized inTable 5. It should be noted that chromosomal origins ofectopic-sequences was established by CASH (Chromosome Assignment usingSomatic cell Hybrids) analysis using the NIGMS Human/Rodent SomaticHybrid Mapping Panel 2 obtained from the Coriell Cell Repositories.Chromosomal assignment was independently confirmed by additional datafor cases pCH1111, pCH172, pCH174, pCH193, and pCH117, as furtheroutlined in Table 5. Taking into account the limitations of conventionalcytogenetic analysis, especially in cases with complex karyotypes, thechromosome assignments of the ectopic sequences are in good agreementwith the previous cytogenetic description of the translocations.

Somewhat unexpected were the data obtained with Ad-312/SV40, asavailable molecular cytogenetic analysis had indicated its chromosome 12breakpoint to map far outside the HMGI-C gene; over 1 Mb [42]. Theectopic sequences appeared to originate from chromosome 1 (moreprecisely from a segment within M.I.T. YAC contig WC-511, which ispartially mapping at 1p22), the established translocation partner (FIG.2). Further molecular analysis is required to precisely define theeffect on functional expression of the aberrant HMGI-C gene in thisparticular case. Of further interest to note here, is that the sequencescoming from chromosome 1 apparently remove the GGGGT repeat observed inthe 3′-UTR region of HMGI-C, as this repeat is not present in the RACEproduct. In contrast, in primary uterine leiomyoma LM-#58(t(8;12)(q24;q14-q15)), which was shown to have its breakpoint also inthe 3′-UTR, this repeat appeared to be present in the RACE product.Therefore, removal of this repeat is most probably not critical fortumor development. The results with primary tumor LM-#168.1, in whichthe X chromosome is the cytogenetically assigned translocation partner,revealed that the ectopic sequences were derived from chromosome 14; thepreferential translocation partner in leiomyoma. It is possible thatinvolvement of chromosome 14 cannot be detected by standard karyotypingin this particular case, as turned out to be the case for Li-501/SV40.In primary lipoma Li-#294 (t(8;12)(q22;q14)), two alternative ectopicsequences were detected. Additional CASH analysis using a hybrid cellmapping panel for regional localization of probes to human chromosome 8[68] revealed that these were both derived from chromosome 8q22-qter(Table 5). It is very well possible that these RACE products correspondto alternatively spliced transcripts. Finally, in four of the cases(Table 5, cases pCH114, pCH110, pCH109, pCH116), the RACE productsappeared to correspond to cryptic or aberrantly spliced HMGI-Ctranscripts, as the corresponding ectopic sequences were found to bederived from either HMGI-C intron 3 or 4. Such RACE products have alsobeen observed in the control experiments described above. In conclusion,the detection of aberrant HMGI-C transcripts in the tumor cells providesadditional strong support of HMGI-C being consistently rearranged by thevarious chromosome 12 aberrations. It should be noted that the aberrantHMGI-C transcripts in the various cases should be characterized in fulllength before any final conclusion can be drawn about biologicalimplications.

A first and preliminary evaluation of isolated ectopic sequencesrevealed in phase open reading frames of variable length. In the case ofprimary tumor LM-#25, for instance, already the second codon in theectopic sequences appeared to be a stop codon (Table 5). A note ofcaution is appropriate here, as sequence data have been obtained onlyfor clones that were produced via two rounds of extensive (probablymutations inducing) PCR. For Li-501/SV40, it is of interest to notethat, in Northern blot analysis, the isolated ectopic sequences detecteda transcript of over 10 kb in a variety of tissues, including heart,kidney, liver, lung, pancreas, placenta, and skeletal muscle, but not inbrain (data not shown). As chromosome 3 is the preferred partner in thechromosome 12q13-q15 translocations in lipomas and the chromosome 3breakpoints of various lipomas were found to be spanned by YAC cloneCEPH192B10, the detected transcript might correspond to a putativelipoma-preferred partner gene (LPP).

4. Discussion

In ANNEX 1 it was demonstrated that the chromosome 12q13-q15 breakpointsof lipoma, pleomorphic salivary gland adenoma, and uterine leiomyoma,irrespective of their cytogenetic assignments in the past to segmentq13, q14, or q15 of chromosome 12, all cluster within the 1.7 Mb DNAinterval designated MAR. In support of the claimed clustering ofbreakpoints is a recent study by Schoenberg Fejzo et al. [14],identifying a CEPH mega-YAC spanning the chromosome 12 translocationbreakpoints in two of the three tumor types. In the present study, wehave conclusively demonstrated by FISH analysis that chromosome 12breakpoints of seven different solid tumor types are clustering within arelatively small (175 kb) segment of MAR. For some tumor cell lines,Southern blot data were obtained and these were always in support of theFISH results. From all these observations, we conclude that this segmentof MAR constitutes a major target area for the chromosome 12 aberrationsin these tumors and that it is likely to represent the postulated MAGlocus: the multi-tumor aberrant growth locus that might be considered ascommon denominator in these tumors.

Within the 175 kb MAR segment, we have identified the HMGI-C gene anddetermined characteristics of its genomic organization. Structurally,the HMGI-C-encoded phosphoprotein consists of three putative DNA bindingdomains, a spacer region, and an acidic carboxy-terminal domain, andcontains potential sites of phosphorylation for both casein kinase IIand p34/cdc2 [65, 67]. We have provided strong evidence that HMGI-C is aprime candidate target gene involved in the various tumor types studiedhere. In FISH studies, the breakpoints of 29 out of 33 primary tumorswere found to be mapping between two highly informative cosmids 142H1and 27E12; the first one containing the three DBD-encoding exons and thesecond one, the remaining exons that code for the two other domains.Therefore, the majority of the breakpoints map within the gene, most ofthem probably within the 140 kb intron (intron 3), which is also in linewith FISH results obtained with the 26 tumor cell lines that wereevaluated. It should also be noted that the 5′-end of the HMGI-C gene isnot yet fully characterized. As HMGI(Y), another member of this genefamily, is known to possess various alternative first exons (69], thesize of the HMGI-C gene might be larger than yet assumed. Furthersupport that HMGI-C is affected by the chromosome 12 aberrations can bededuced from the results of the 3 ′-RACE experiments. Aberrant HMGI-Ctranscripts were detected in tumor cells, consisting of transcribedHMGI-C sequences fused to newly acquired sequences, in most casesclearly originating from the chromosomes that were cytogeneticallyidentified as the translocation partners. It is noteworthy that manychromosomes have been found as translocation partner in the tumorsstudied. This observed heterogeneity in the reciprocal breakpointregions involved in these translocations resembles that of a variety ofhematological malignancies with chromosome 11q23 rearrangementsinvolving the MLL gene [70], the translational product of which carriesan amino-terminal motif related to the DNA-binding motifs of HMGIproteins.

An intriguing issue pertains to the effect of the chromosome 12aberrations on expression of the HMGI-C gene and the directphysiological implications. Some functional characteristics of HMGI-Care known or may be deduced speculatively from studies of other familymembers. As it binds in the minor groove of DNA, it has been suggestedthat HMGI-C may play a role in organising satellite chromatin or act asa transcription factor (71, 72]. Studies on HMGI(Y), which is the membermost closely related to HMGI-C, have suggested that HMGI(Y) may functionas a promoter-specific accessory factor for NF-κ B transcriptionalactivity [73]. MGI(Y) has also been shown to stimulate or inhibit DNAinding of distinct transcriptional factor ATF-2 isoforms [74]. Bothstudies indicate that the protein may simply constitute a structuralcomponent of the transcriptional apparatus functioning inpromoter/enhancer contexts. In a recent report on high mobility groupprotein 1 (HMG1), yet another member of the HMG gene family with asimilar domain structure as HMGI-C and acting as a quasi-transcriptionfactor in gene transcription, a truncated HMG1 protein lacking theacidic carboxy-terminal region was shown to inhibit gene transcription[75]. It was put forward that the acidic terminus of the HMG1 moleculeis essential for the enhancement of gene expression in addition toelimination of the repression caused by the DNA binding. As most of thechromosome 12 breakpoints seem to occur in the 140 kb intron, separationof the DBDs from the acidic carboxy-terminal domain seems to occurfrequently. In case the acidic domain in HMGI-C has a similar functionas the one in HGMI(Y), the result of the chromosome 12 aberrations islikely to affect gene expression. Finally, it should be noted that thefate of the sequences encoding the acidic carboxy-terminal region is notyet known. As HMGI-C is the first member of the HMG gene family thatmight be implicated in the development of benign tumors, the questionarises as to whether other members of this family could also beinvolved. The HMG protein family consists of three subfamilies: i) theHMG1 and 2 type proteins, which have been found to enhance transcriptionin vitro and may well be members of a much larger class of regulatorswith HMG boxes; ii) the random-coil proteins HMG14 and 17 with an as yetunknown function; iii) the HMGI-type proteins, which bind to the minorgroove and include HMGI-C, HMGI, and HMGI-Y; the latter two are encodedby the same gene. It is of interest to note that published mappingpositions of members of the HMG family coincide with publishedchromosome breakpoints of benign solid tumors such as those studiedhere. The HMGI(Y) gene, for instance, has been mapped to humanchromosome 6p21 [69], which is known to be involved in recurrenttranslocations observed in uterine leiomyoma, lipoma, and pleomorphicsalivary gland adenoma [76]. As listed in the Human Genome Data Base,not all known members of the HMG family have been chromosomally assignedyet, although for some of them a relatively precise mapping position hasbeen establisher. For instance, HMG17 to chromosome 1p36.1-p35, HMG1L to13q12, and HMG14 to 21q22.3; all chromosome segments in which chromosomebreakpoints of the tumor types studied here have been reported [76].Whether HMGI(Y) or any other of these HMG members are indeed affected inother subgroups of these tumors remains to be established. Of interestto mention, furthermore, are syndromes such as Bannayan-Zonana (McKusick#153480), Proteus (McKusick #176920), and Cowden (McKusick #158350); thelatter syndrome is also called multiple hamartoma syndrome. In 60% ofthe individuals with congenital Bannayan-Zonana syndrome, a familialmacrocephaly with mesodermal hamartomas, discrete lipomas andhemangiomas were found [70].

Finally, one aspect of our results should not escape attention. All thetumors that were evaluated in this study were of mesenchymal origin orcontained mesenchymal components. It would be of great interest to findout whether the observed involvement of HMGI-C is mesenchyme-specific ormay be found also in tumors of non-mesenchymal origin. The various DNAclones we describe here are valuable resources to address this importantissue and should facilitate studies to conclusively implicate the HMGI-Cgene in tumorigenesis.

Example 3 Rearrangements of Another Member of the HMG Gene Family

1. Introduction

This example clearly demonstrates that within a given tumor entity (e.g.pulmonary chondroid hamartomas, uterine leiomyomas, endometrial polyps)tumors, histologically practically indistinguishable from each other,arise if either the HMGI-C gene or the HMGI(Y) gene is affected bychromosomal rearrangements. Thus, indeed a group of genes leading toaberrant mesenchymal growth including but not restricted to HMGI-C andHMGI(Y) can be defined.

2. Material and Methods

2.1. Chromosome Preparation

Chromosome preparation followed routine methods. Cells were treated with30 μl colcemide (10 μg/ml) for 2-3 h and then harvested using thetrypsin method (0.05% trypsin, 0.02% EDTA) followed by a hypotonic shockin six fold diluted medium TC 199 for 20 minutes at room temperature andmethanol:acetic acid (3:1) fixation. Chromosomes were then GTG-banded.

2.2. In situ Hybridization

In situ hybridisation was performed as outlined in one of the previousexamples.

2.3. PAC Library Screening

The PAC library (Genome Systems Library Screening Service, St. Louis,Mo., USA) was screened by PCR with a primer set specific for the HMGI(Y)gene. For screening we designed the forward primer with the sequence:

5′-CTC CAA GAC AGG CCT CTG ATG T-3′ (intron 3)(SEQ ID No:9) and thereverse primer:

5′-ACC ACA GGT CCC CTT CAA ACT A-3′ (intron 3)(SEQ ID No:10) giving riseto a fragment of 338 bp. For amplification the following thermal cyclingwas used: 94° C., 5 min, (94° C., 1 min, 59° C., 1 min, 72° C., 2min)×30, 72° C., 10 min.

2.4. DNA Preparations From PAC Clones

Bacterial colonies containing single PAC clones were inoculated into LBmedium and grown overnight at 37° C. 660 μl of the overnight culturewere diluted into 25 ml of LB medium and grown to an OD₅₅₀ of 0.05-0.1.By addition of IPTG to a final concentration of 0.5 mM the P1 lyticreplicon was induced. After addition of IPTG, growth was continued to anOD₅₅₀ of 0.5-1.5, and plasmid DNA was extracted using the alkaline lysisprocedure recommended by Genome Systems.

3. Results

The primer set for screening the human PAC library was designed fromsequences belonging to intron 3 of HMGI(Y). Because of sequence homologybetween HMGI-C and HMGI(Y) the amplified sequence of 338 bp was testedby homology search to be specific exclusively for HMGI(Y). Libraryscreening resulted in three positive PAC clones that had an averageinsert length of approximately l00 kb. Two of these clones (Pac604,Pac605) were used for the following FISH studies. In order to prove ifHMGI(Y) is rearranged in tumors with translocations involving 6p21.3 ineither simple or complex form we performed FISH analysis on metaphasespreads from four primary pulmonary chondroid hamartomas and twoendometrial polyps all with 6p21.3 abnormalities. For each case 20metaphases were scored. At least one of the two PAC clones Pac604 andPac605 described above was across the breakpoint in all six casesanalyzed. These results clearly show that the breakpoints of the tumorswith 6p21 aberrations investigated herein are clustered either withinthe HMGI(Y) gene or its close vicinity.

Example 4 Hybrid HMGI-C in Lipoma Cells

CDNA clones of the chromosome 3-derived lipoma-preferred partner geneLPP (>50 kb) were isolated and the nucleotide sequence thereofestablished. Data of a composite cDNA are shown in FIG. 4. An openreading frame for a protein (612 amino acids (aa)) with amino acidsequence similarity (over 50%) to zyxin of chicken was identified. Zyxinis a member of the LIM protein family, whose members all possessso-called LIM domains [78]. LIM domains are cysteine-rich, zinc-bindingprotein sequences that are found in a growing number of proteins withdivers functions, including transcription regulators, proto-oncogeneproducts, and adhesion plaque constituents. Many of the LIM familymembers have been postulated to play a role in cell signalling andcontrol of cell fate during development. Recently, it was demonstratedthat LIM domains are modular protein-binding interfaces [79]. Likezyxin, which is present at sites of cell adhesion to the extracellularmatrix and to other cells, the deduced LPP-encoded protein (FIG. 6)possesses three LIM domains and lacks classical DNA-bindinghomeodomains.

In 3′-RACE analysis of Li-501/SV40, a HMG1-C containing fusiontranscript was identified from which a hybrid protein (324 aa) could bepredicted and which was subsequently predicted to consist of the threeDBDs (83 aa) of HMG1-C and, carboxy-terminally of these, the three LIMdomains (241 aa) encoded by LPP. In PCR analysis using approriate nestedamplimer sets similar HMGI-C/LPP hybrid transcripts were detected invarious primary lipomas and lipoma cell lines carrying a t(3;12) andalso in a cytogenetically normal lipoma. These data reveal that thecytogenetically detectable and also the hidden t(3;12) translocations inlipomas seem to result consistently in the in-phase fusion of theDNA-binding molecules of HMG1-C to the presumptive modularprotein-binding interfaces of the LPP-encoded protein, thereby replacingthe acidic domain of HMG1-C by LIM domains. Consequently, theseprotein-binding interfaces are most likely presented in the nuclearenvironment of these lipoma cells, where they might affect geneexpression, possibly leading to aberrant growth control. Out of thelarge variety of benign mesenchymal tumors with chromosome 12q13-q15aberrations, this is the first example of a chromosome translocationpartner contributing recurrently and consistently to the formation of awell-defined tumor-associated HMG1-C fusion protein.

FIG. 5 shows the cDNA sequence of the complete isolated LPP gene.

Example 5 Diagnostic Test for Lipoma

A biopsy of a patient having a lipoma was taken. From the material thusobtained total RNA was extracted using the standard TRIZOL™ LS protocolfrom GIBCO/BRL as described in the manual of the manufacturer. Thistotal RNA was used to prepare the first strand of CDNA using reversetranscriptase (GIBCO/BRL) and an oligo dT(17) primer containing anattached short additional nucleotide stretch. The sequence of the primerused is as described in Example 2, under point 2.5. RNase H wassubsequently used to remove the RNA from the synthesized DNA/RNA hybridmolecule. PCR was performed using a gene specific primer (Example 2,point 2.5. ) and a primer complementary to the attached short additionalnucleotide stretch. The thus obtained PCR product was analysed by gelelectroforesis. Fusion constructs were detected by comparing them withthe background bands of normal cells of the same individual.

In an additional experiment a second round of hemi-nested PCR wasperformed using one internal primer and the primer complementary to theshort nucleotide stretch. The sensitivity of the test was thussignificantly improved.

FIG. 8 shows a typical gel.

Example 6 Aberrations of 12q14-15 and 6p21 in Pulmonary ChondroidHamartomas

1. Introduction

Pulmonary chondroid hamartomas (PCH) are often detected during X-rayexamination of the lung as so-called coin lesions. However, lungmetastases of malignant tumors and rarely lung cancers can also presentas coin lesions. This example shows that FISH requiring a minimal amountof tumor cells can be used to correctly distinguish between the majorityof PCHs and malignant tumors. Thus the test can successfully be appliede.g. to tumor cells obtained by fine needle aspiration.

2. Materials and Methods

Samples from a total of 80 histologically characterized PCHs wereincluded in this study. Cell cultures, chromosome preparations and FISHwere obtained or performed as described in the previous examples.

3. Results

Cytogenetic studies revealed that of the 80 PCHs studied cytogenetically51 revealed detectable aberrations involving either 12q14-15 or 6p21. ByFISH using either a pool of cosmids belonging to the HMGI-C gene orusing the PAC clones of HMGI(Y) described in the previous example wewere able to detect hidden structural rearrangements of those regions in4 additional cases (3 with 12q and one with 6p involvement). Therefore,the FISH test alone can be used for a kit to precisely detect therearrangement of either the HMGI-C or the HMGI(Y) gene rearrangements inmore than 50% of the PCHs and is thus a valuable additonal tool for thediagnosis of these tumors (without being restricted to this type oftumors as shown in two of the other examples).

Example 7 Diagnosis of Soft Tissue Tumors Particularly of AdipocyticOrigin

1. Introduction

Adipocyte tissue tumors often cause diagnostic difficulties particularlywhen material taken from fine needle aspiration biopsies or cryosectionshas to be evaluated. This examples demonstrates the validity of the FISHtest for the differential diagnosis of adipocyte tissue tumors and raresoft tissue tumors.

2. Materials and Methods

2.1. Tumor Samples

Tumor samples from three soft tissue tumors were investigated by FISH.Sample one (1) was from a adipocytic tumor and histologically it waseither an atypical lipoma or a well-differentiated liposarcoma. Thesecond case (tumor 2) was diagnosed to be most likely a myxoidliposarcoma but other types of malignant soft tissue tumors includingaggressive angiomyxoma were also considered. The third tumor (tumor 3)was also of adipocytic origin and both a lipoma and a welldifferentiated liposarcoma were considered.

2.2. Isolation of Cells and FISH

The tumor samples were enzymatically disaggregated following routinemethods. The resulting single cell suspensions were centrifuged and thesuspensions were fixed using methanol:glacial acetic acid (3:1) at roomtemperature for 1 hour. The cell suspensions were then dropped on cleandry slides and allowed to age for 6 hours at 60° C. FISH was performedusing molecular probes from the HMGI-C gene as described in the previousexamples.

3. Results

At the interphase level tumor 1 and 2 both showed split signals for oneof the alleles. These findings are compatible with the diagnosis ofbenign tumors i.e. an atypical lipoma in the first case and anaggressive angiomyxoma in the second case. They allowed to rule out thepresence of malignant adipocytic tissue tumors.

In the third case the FISH revealed a high degree of amplification ofthe MAR region or part of it. Since the amplification units observed ingiant marker or ring chromosomes in well-differentiated liposarcomas caninvolve the MAR region these findings leads to the diagnosis of awell-differentiated liposarcoma. The three cases presented within thisexample show the usefulness of the DNA probes described. They can beused in a kit for a relatively simple and fast interphase FISHexperiment offering an additional tool for the diagnosis of soft tissuetumors.

Example 8 Expression of the HMGI-C Gene in Normal Tissue

1. Introduction

It is the aim of this example to show that the expression of the HMGI-Cgene is mainly restricted to human tissues during embryonic and fetaldevelopment. In contrast, in most normal tissues of the adultparticularly, including those tissues and organs tumors with HMGI-Crearrangements can arise from, on expression can be noted. Thisindicates that even the transcriptional re-activation of the gene caninititate tumorigenesis. On the other hand it underlines the usefulnessof antisense strategies (including those antisense molecules directedtowards the normal HMGI-C mRNA) to inhibit or stop tumor growth.

2. Materials and Methods

2.1. Tissue Samples

All adult tissue samples used for this study were taken from surgicallyremoved tissue frozen in liquid nitrogen within a period of 15 min afterremoval. Most of the samples were from adjacent normal tissue removedduring tumor surgery. In detail we have used 8 samples taken from fattissues at various anatomical sites, 20 samples taken from myometrialtissue, 8 samples taken from lung tissue, 4 samples taken from thesalivary glands (Glandula parotis and Glandula submandibularis), onetissue sample taken from the heart muscle, 25 samples taken from breasttissue from patients of different ages, 2 samples from the brain, 3liver samples, 7 samples taken from renal tissue, and embryonic/fetaltissue (extremeties, 6 samples) from embryos/fetuses (10-14thgestational week) after abortion from socio-economic reasons.

In addition, three cell lines were used: As a control for HMGI-Cexpression we used the hepatoma cell line Hep 3B and the cell line L14established from a lipoma with the typical translocation t(3;12). HeLacells were used as a negative control because RT experiments reproducedfor 10 times did not reveal HMGI-C expression in our own studies.

2.2. RT-PCR for the Expression of HMGI-C

100 mg of tissue sample was homogenized, and RNA was isolated using thetrizol reagent (GibcoBRL, Eggenstein, Germany) containing phenol andisothiocyanate. cDNA was synthesized using a poly(A)-oligo(dt)17 primerand M-MLV reverse transcriptase (GibcoBRL, Eggenstein, Germany). Then, ahemi-nested PCR was performed.

For first and second PCR the same lower primer (Revex 4) (5′-TCC TCC TGAGCA GGC TTC-3′ (exon 4/5))(SEQ ID No:11) was used. In the first round ofPCR the specific upper primer (SE1) (5′-CTT CAG CCC AGG GAC AAC-3′ (exon1)(SEQ ID No:12), and in the second round of PCR the nested upper primer(P1) (5′-CGC CTC AGA AGA GAG GAC-3′ (exon 1)(SEQ ID No:13) was used.Both rounds of PCR were performed in a 100 μl volume containing 10 mMTris/HCl pH 8.0, 50 mM KCl, 1.5 mM MgCl₂, 0.001% gelatin, 100 μM DATP,100 μM dTTP, 100 μM dGTP, 100 μM dCTP, 200 nM upper primer, 200 nM lowerprimer, and 1 unit/100 μl AmpliTaq polymerase (Perkin Elmer,Weiterstadt, Germany). Amplification was performed for 30 cycles (1 min94° C., 1 min 53° C., 2 min 72° C.). As template in the first round ofPCR cDNA derived from 250 ng total RNA, and in the second round of PCR 1μl of the first PCR reaction mix was used.

2.3. Control Assay for Intact mRNA/cDNA

As control reaction for intact RNA and cDNA PCR a test based on theamplification of the cDNA of the housekeeping gene glyceraldehyde3-phosphate dehydrogenase (GAPDH). PCR reaction was performed for 35cycles under the same conditions as described above for the first roundof PCR of HMGI-C expression.

3. Results

As for the expression studies all experiments were repeated at leasttwice. To assure that all RNA and cDNA preparations used for the RT-PCRswere intact (otherwise resulting in false negative results) routinely aRT-PCR for expression of the housekeeping gene GAPDH was performed. Apositive GAPDH RT-PCR results in a 299 bp fragment. Only samplesrevealing a positive GAPDH RT-PCR were included in this study. As theresult of RT-PCR in HMGI-C positive cells such as Hep 3B and L14 aspecific 220 bp fragment is detectable. HeLa cells did not show anexpression of HMGI-C. Except for two myometrial samples (most likely dueto myomas at a submicroscopic level) all normal tissue samples takenfrom adult individuals did not show any detectable level of HMGI-Cexpression. In contrast, all fetal/embryonic tissues tested revealedHMGI-C expression.

Example 9 Expression of the HMGI-C Gene as a Diagnostic Tool for theEarly Detection of Leukemias

1. Introduction

Cytogenetically detectable aberrations affecting the HMGI-C gene havebeen found in a variety of benign solid tumors of mesenchymal origin.Apparently, the aberrations also lead to the transcriptional activationof the gene. Since blood cells are also of mesenchymal origin, it wastempting to check leukemic cells for HMGI-C expression. The presentexample shows that the activation of the gene in cells of the peripheralblood is a suitable marker indicating immature cells/abnormal stem cellsfound in leukemias. Since the expression of HMGI-C can be determinedwith a high degree of sensitivity the RT-PCR for the expression of thegene can be used for a very early detection of various hematologicaldiseases.

2. Materials and Methods

Samples from peripheral blood of 27 patients with different types ofleukemias including 19 patients with Philadelphia-chromosome positiveCML, 5 patients with AML, and 3 patients with ALL were used fordetermination of HMGI-C expression. Blood samples from 15 healthyprobands served as controls.

RT-PCR for the expression of HMGI-C was performed as outlined in example8.

3. Results

Whereas expression of HMGI-C was clearly detectable in all blood samplesfrom leukemic patients there was no expression noted in any of the bloodsamples taken from the control persons. There is no evidence that thetranscriptional activation of the gene is due to mutations affecting thegene or its surroundings. It is more reasonable to assume that theactivation is rather a secondary effect related to the immaturity of thecells or their abnormal proliferation. However, the high and evenimprovable sensitivity makes e.g. a kit based on the RT-PCR for theexpression of the HMGI-C gene a very suitable diagnostic tool.

Example 10 The Transcriptional Re-expression of the HMGI-C Gene can Leadto the Initiation of the Tumors

1. Introduction

This example clearly shows that for some tumor entities chromosomalbreakpoints located 5′ of the HMGI-C gene do also exist indicating thatthe transcriptional up-regulation of the gene is sufficient to initiategrowth of the corresponding tumor types.

2. Materials and Methods

2.1. Cell Culture

After surgery the tumor samples (three pulmonary chondroid hamartomas,one uterine leiomyoma) were washed with Hank's solution supplementedwith penicillin (200 IU/ml) and streptomycin (200 μg/ml). Tumors weredisaggregated with collagenase for 5-6 h at 37° C. The suspensioncontaining small fragments and single cells was resuspended in culturemedium TC 199 with Earle's salts supplemented with 20% fetal bovineserum, 200 IU/ml penicillin, and 200 μg/ml streptomycin.

2.2. Chromosome Preparations

Chromosome preparation followed routine methods. Cells were treated with30 μl colcemide (10 μg/ml) for 2-3 h and then harvested using thetrypsin method (0.05% trypsin, 0.02% EDTA) followed by a hypotonic shockin six fold diluted medium TC 199 for 20 minutes at room temperature andmethanol:acetic acid (3:1) fixation. Chromosomes were then GTG-banded.

2.3. FISH Studies

To identify the chromosomes unambiguously, FISH was performed afterGTG-banding of the same metaphase spreads. As DNA probes we used fivecosmids belonging to a YAC-contig overspanning the HMGI-C gene asdescribed in the Legenda of FIG. 2. Three of these cosmids (27E12,185H2, 142H1) are mapping to the third intron of HMGI-C, whereas cosmids260C7 and 245E8 are localized at the 3′ or the 5′ end respectively. Theslides were analyzed using a Zeiss (Zeiss, Oberkochem, Germany) Axioplanfluorescence microscope. Results were processed and recorded with thePower Gene Karyotyping System (PSI, Halladale, Great Britain). Rapidamplification of cDNA ends (RACE) was performed as described in one ofthe former examples.

3. Results

All four tumors showed the same type of cytogenetic abnormality, i.e.the presence of 47 chromosomes including two apparently normalchromosomes 12 and an additional derivative 14der(14)t(12;14)(q14-15;q24) but without a corresponding der(12). Sincethe 3′-55′ orientation of the HMGI-C is towards the centromere a singlebreak within the HMGI-C gene would have led to the loss of its 5′ partalong with the loss of the der(12). We have therefore performed a seriesof FISH experiments in order to determine the breakpoints moreprecisely. Using the five cosmids 260C7, 27E12, 185H2, 142H1, and 245E8hybridization signals of the same intensity were observed at both normalchromosomes 12 and at the additional der(14). The FISH results revealedthat in all four cases chromosomal breakpoints were located 5′ of theHMGI-C gene.

The breakpoint assignment in all four cases 5′ of the HMGI-C gene fitswell with the results of the RACE-PCR. In addition to the normal HMGI-Ctranscripts we were able to detect aberrant transcripts in all threetumors. Sequences showed that they were not derived from chromosome 14but from intron 3 of HMGI-C probably due to cryptic splice sites.However, the RACE results revealed that there was indeed HMGI-Cexpression in all four cases.

Example 11 Re-differentiation of Leukemic Cells

1. Introduction

Expression of the HMGI-C gene is frequently strongly elevated in a widevariety of tumors, solid tumors as well as leukemias. It was speculatedthat the HMGI-C protein might play a key role in transformation ofcells. This example shows that expression of the HMGI-C gene can bestrongly reduced by expressing antisense HMGI-C sequences and thatreduction of HMGI-C levels in tumor cells results in reversion of thetransformed phenotype. Thus the expression or administration ofantisense molecules can be successfully applied therapeutically.

2. Materials and Methods

2.1. Tumor Cell Lines

Tumor cell lines were generated from a primary malignant salivary glandtumor and a primary breast carcinoma. Cell lines were established asdescribed by Kazmierczak, B., Thode, B., Bartnitzke, S., Bullerdiek, J.and Schloot, W., “Pleomorphic adenoma cells vary in their susceptibilityto SV40 transformation depending on the initial karytotype.”, GenesChrom. Cancer 5:35-39 (1992).

2.2. Assay of the Transformed State

Soft agar colony assays were performed as described by Macpherson andMontagnier, “Agar suspension culture for the selective assays of cellstransformed by polyoma virus.” Virology 23, 291-294 (1964).

Salivary gland and breast tumor cells were propagated in TC199 culturemedium with Earle's salts, supplemented with 20% fetal bovine serum(GIBCO), 200 IU/ml penicillin, and 200 μg/ml streptomycin.

Tumorigenicity of the transfected salivary gland (AD64) and breast celllines was tested by injecting cells subcutaneously into athymic mice.

2.3. Transfection Assay

Transfections were performed using various protocols, namely:

1. The calcium phosphate procedure of Graham and Van der Eb (“A newtechnique for the assay of the infectivity of human adenovirus.”Virology 52, 456-467 (1973) ).

2. Lipofection: Transfections were carried out using liposome-mediatedDNA transfer (lipofectamine, GibcoBRL) according to the guidelines ofthe manufacturer.

2.4. Antisense Constructs

Sense and antisense constructs of the HMGI-C gene were obtained byinserting human HMGI-C CDNA sequences in both the sense and antisenseorientation in expression vectors under the transcriptional control ofvarious promoter contexts, e.g. the long terminal repeat of Moloneymurine leukemia virus, a CMV promoter, or the early promoter of SV40.For example, the CMV/HMGI-C plasmid was constructed by cloning a humanHMGI-C cDNA fragment containing all coding sequences of human HMGI-C inpRC/CMV (Invitogen) allowing expression under control of the humancytomegalovirus early promoter and enhancer, and selection for G418resistance.

3. Results

3.1. Reversion of the Transformed Phenotype

Reversion of the transformed phenotype was observed in breast andsalivary gland tumors cells after induction of antisense HMGI-Cexpression in these tumor cells. A strong reduction in tumorigenicitywas observed as measured by soft agar colony assay and in vivo inathymic mice. Immunoprecipitation and Western blot analysis indicated astrong reduction of HMGI-C protein levels in the cells expressingantisense HMGI-C sequences. Therefore, this approach can be usedtherapeutically in tumors with involvement of HMGI-C.

Example 12 Animal Tumor Models Involving HMGI-C as Tools in in vivoTherapeutic Drug Testing

On the basis of the acquired HMGI-C knowledge, animal tumor models canbe developed as tools for in vivo drug testing. To achieve thisobjective (for instance for uterine leiomyoma), two approaches can beused, namely gene transfer (generation of transgenic animals) on the onehand and gene targeting technology (mimicking in vivo of a specificgenetic aberration via homologous recombination in embryonic stem cells(ES cells)) on the other.

These technologies allow manipulation of the genetic constitution ofcomplex living systems in specific and pre-designed ways. For extensivetechnical details, see B. Hogan, R. Beddington, F. Constantini, and E.Lacy; In: Manipulating the mouse embryo, A Laboratory Manual. ColdSpring Harbor Press, 1994; ISBN 0-87969-384-3.

To aim at the inactivation or mutation of the HMGI-C gene, specificallyin selected cell types and selected moments in time, the recentlydescribed Cre/LoxP system can be used (Gu, H. et al. Deletion of a DNApolymerase β gene segment in T cells using cell type-specific genetargeting. Science 265, 103-106, 1994). The Cre enzyme is a recombinasefrom bacteriophage P1 whose physiological role is to separate phagegenomes that become joined to one another during infection. To achieveso, Cre lines up short sequences of phage DNA, called loxP sites andremoves the DNA between them, leaving one loxP site behind. This systemhas now been shown to be effective in mammalian cells in excising athigh efficiency chromosomal DNA. Tissue-specific inactivation ormutation of a gene using this system can be obtained via tissue-specificexpression of the Cre enzyme.

As an example, the development of animal model systems for uterineleiomyoma using a member of the MAG gene family will be outlined below,such that the models will be instrumental in in vivo testing oftherapeutic drugs.

Two approaches may be followed:

a) in vivo induction of specific genetic aberrations as observed inhuman patients ((conditional) gene (isogenic) targeting approach); and

b) introduction of DNA constructs representative for the geneticaberrations observed in patients (gene transfer approach).

DNA constructs to be used in gene transfer may be generated on the basisof observations made in patients suffering from uterine leiomyoma as faras structure and expression control are concerned; e.g. HMGI-C fusiongenes with various translocation partner genes, especially thepreferential translocation partner gene of chromosome 14 located in theYAC contig represented by CEPH YACs 6C3, 89C5, 308H7, 336H12, 460A6,489F4, 902F10, 952F5, 958C2, 961E1, and 971F5, truncated genes encodingbasically the three DNA binding domains of HMGI-C, and complete HMGI-Cor derivatives of HMGI-C under control of a strong promoter.

Example 13 The Preparation of Antibodies Against HMGI-C

One type of suitable molecules for use in diagnosis and therapy areantibodies directed against the MAG genes. For the preparation of rabbitpolyclonal antibodies against HMGI-C use was made of the following threecommercially available peptides:

(H-ARGEGAGQPSTSAQGQPAAPAPQKR) 8-Multiple Antigen Peptide (SEQ ID No:14)

(H-SPSKAAQKAEATGEK)8-MAPA (SEQ ID No: 15)

(H-PRKWPQQVVQKKPAQEE)8-MAP (SEQ ID No:16)

obtainable from Research Genetics Inc., Huntsville, Ala., USA. Thepolyclonal antibodies were made according to standard techniques.

TABLE I ANALYSIS OF YAC CLONES CEPH-Code Size (kb) Landmark left #Landmark right # Chimeric 183F3 715 [RM10] YES (L + R) 70E1 450 RM29U27125 ND 95F1 390 RM30 U29054 ND 201H7 320 RM23 U29051 RM14 U29053 ND185G12 320 ND 354B6 280 YES (R) 126G8 410 ND 258F11 415 RM4 U29052 ND320F5 290 RM5 U29050 RM21 U29047 ND 234G11 475 RM7 U29046 ND 375H5 290ND 262E10 510 [RM15] RM16 U29048 YES (L) 181C8 470 RM26 U29045 ND 107D1345 RM31 U29043 Nb 499C5 320 RM44 U29044 RM46 U29337 ND 340B6 285 ND532C12 400 RM45 U29041 ND 138C5 510 [RM59] RM65 U29342 YES (L) 145F2 490RM60 U29030 RM66 U29340 ND 106E8 340 RM57 U29033 RM63 U29038 ND 55G1 365RM56 U29031 RM62 U29039 ND 103G7 370 RM85 U29025 RM60 U29335 ND 295B10295 RM77 U29035 RM61 U29026 ND 338C2 200 RM78 U29034 RM82 U29029 ND391C12 160 [RM79] RM83 U29027 YES (L) 476A11 225 [RM87] RM84 U25032 YES(L) 138F3 460 RM90 U29028 RM91 U29019 ND 226E7 500 RM48 U29024 RM54U29015 ND 499E9 375 RM51 U29016 YES (R) 312F10 580 [RM50] RM69 U29021YES (L) 825G7 950 ND 34B5 315 RM88 U29020 RM89 U29013 ND 94A7 610 YES(R) 305B2 660 YES (L) 379H1 280 RM204 U29014 RM105 U29009 ND 444E6 350RM92 U29017 RM93 U29010 ND 446H3 370 RM94 U29011 RM95 U29018 ND 403B12380 ND 261E5 500 RM102 U29012 RM103 U26689 ND 78B11 425 ND 921B9 1670 ND939H2 1750 ND 188H7 360 ND 142F4 390 ND 404E12 360 ND 164A3 375 ND244B12 415 RM106 U29307 RM107 U29008 ND 275H4 345 RM108 U29004 RM109U29005 ND 320F9 370 ND 51F8 450 ND 242A2 160 CH1 U29005 ND 253H1 400 ND303F11 320 ND 322C8 410 CH2 U29003 ND 208G12 370 RM96 U29002 RM97 U27125ND 341C1 270 RM98 U26847 RM99 U27130 ND 354F1 270 ND 452E1 270 CH5U27136 ND 41A2 310 ND 934D2 1370 ND 944E8 1290 CH8 U23792 ND 2G11 350 ND755D7 1390 YES (L) 365A12 370 ND 803C2 1080 ND 210C1 395 RM70 U28998RM86 U27133 ND 433C8 360 RM73 U29000 RM76 U27132 ND 402A7 500 RM41U28994 [RM42] YES (R) 227E8 465 RM53 U27134 RM55 U28995 ND 329F9 275RM72 U28793 RM75 U28997 ND 261E5 395 [RM71] RM74 U28995 YES (L) 348F2370 [RM136] YES (R) 6F3 320 RM35 U27140 RM36 U27141 59F12 430 RM34U26794 RM33 U27131 255H3 300 RM40 U28999 YAC clonees were isolated fromCEPH YAC libraries as described in Materials and Methods. ND: notdetected by methods used. Landmarks not mapping within the 6 Mb contighave been bracketed. GenBank accession numbers are given (#).

TABLE II PCR Primers (SEQ ID Nos: 17-18) STS name Nucleotide Productsize T_(ann) (STS 12-) sequence 5′-3′ (bp) (° C.) CH1TGGGACTAACGGATTTTCAA 213 58 TGTGGTTCATTCATGCATTA CH2TCCATCATCATCTCAAAACA 145 58 CTCTACCAAATGGAATAAACAG CH5GCAGCTCAGGCTCCTTCCCA 143 58 TGGCTTCCTGAAACGCGAGA CH8TCTCCACTGCTTCCATTCAC 147 58 ACACAAAACCACTGGGGTCT CH9CAGCTTTGGAATCAGTGAGG 262 58 CCTGGGGAAGAGGAGTAAAG RM1 GAGCTTCCTATCTCATCC308 60 ATGCTTGTGTGTGAGTGG RM4 TTTGCTAAGCTAGGTGCC 236 60AGCTTCAAGACCCATGAG RM5 CAGTTCTGAGACTGCTTG 324 60 TAATAGCAGGGACTCAGC RM7CTTGTCTCATTCTTTTAAAGGG 538 58 CACCCCTTTTTAGATCCTAC RM13GAATGTTCATCACAGTGCTG ±500 58 AATGTGAGGTTCTGCTGAAG RM14TTCTCATGGGGTAAGGACAG 158 58 AAAGCTGCTTATATAGGGAATC RM16CCTTGGCTTAGATATGATACAC 252 58 GCTCTTCAGAAATATCCTATGG RM21CCTTAGCAGTTGCTTGTCTG 290 58 TCGTCACAGGACATAGTCAC RM26TCTATGGTATGTTATACAAGATG 102 58 CAGTGAGATCCTGTCTCTA RM31TCTGTGATGTTTTAAGCCACTTAG 239 56 AATTCTGTGTCCCTGCCACC RM33ATTCTTCCTCACCTCCCACC ±600 60 AATCTGCAGAGAGGTCCAGC RM34AATTCTCCATCTGGGCCTGG ±600 60 GAACGCTAAGCATGTGGGAG RM36CTCCAACCATGGTCCAAAAC 296 60 GACCTCCAGTGGCTCTTTAG RM46ACCATCAGATCTGGCACTGA 241 57 TTACATTGGAGCTGTCATGC RM48TCCAGGACATCCTGAAAATG 391 58 AGTATCCTGCACTTCTGCAG RM51GATGAACTCTGAGGTGCCTTC 311 60 TCAAACCCAGCTTTGACTCC RM53GTCTTCAAAACGCTTTCCTG 333 60 TGGTTTGCATAATGGTGATG RM60TACACTACTCTGCAGCACAC 94 58 TCTGAGTCAATCACATGTCC RM69CTCCCCAGATGATCTCTTTC 236 58 CGGTAGGAAATAAAGGAGAG RM72TATTTACTAGCTGGCCTTGG 101 62 CATCTCAGGCACACACAATG RM76ATTCAGAGAAGTGGCCAAGT 496 58 GGGATAGGTCTTCTGCAATC RM85TCCAACAATACTGAGTGACC 435 58 TCCATTTCACTGTAGCACTG RM86GTAATCAACCATTCCCCTGA 203 56 AAAATAGCTGGTATGGTGGC RM90ACTGCTCTAGTTTTCAAGGA 257 58 AATTTACCTGACAGTTTCCT RM93GCATTTGACGTCCAATATTG 347 60 ATTCCATTGGCTAACACAAG RM98GCAAAACTTTGACTGAAACG 356 58 CACAGAGTATCGCACTGCAT RM99AAGAGATTTCCCATGTTGTG 240 58 CTAGTGCCTTCACAAGAACC RM103AATTCTTGAGGGGTTCACTG 199 60 TCCACACTGAGAGCTTTTCA RM108GTGGTTCTGTACAGCAGTGG 439 60 TGAGAAAATGTCTGCCAAAT RM110GCTCTACCAGGCATACAGTG 328 58 ATTCCTAGCATCTTTTCACG RM111ATATGCATTAGGCTCAACCC 312 58 ATCCCACAGGTCAACATGAC RM130ATCCTTACATTTCCAGTGGCATTCA 336 58 CCCAGAAGACCCACATTCCTCAT RM131TTTTAAGTTTCTCCAGGGAGGAGAC 226 58 AATAGGCTCTTTGGAAAGCTGGAGT RM132TCTCAGCTTAATCCAAGAAGGACTTC 376 58 GGCATATTCCTCAACAATTTATGCTT RM133TGGAGAAGCTATGGTGCTTCCTATG 225 58 TGACAAATAGGTGAGGGAAAGTTGTTAT EST01096TCACACGCTGAATCAATCTT 188 58 CAGCAGCTGATACAAGCTTT IFNGTGTTTTCTTTCCCGATAGGT 150 52 CTGGGATGCTCTTCGACCTC Rap1BCCATCCAACATCTTAAATGGAC 149 58 CAGCTGCAAACTCTAGGACTATT STSs were isolatedas described in Materials and Methods, or retrieved from literature forEST01096, IFNG, and Rap1B.

TABLE 3 Genome Data Base accession numbers (D-numbers) of the varioussequences indicated in FIG. 1. Genome Data Base (41 rows affected) perlocus_symbol per per_gdb_id locus locus_gdb_id CH1-lower/CH1-upperD12S1484 CH1-lower/CH1-upper G00-595-292 D12S1484 G00-595-415CH2-lower/CH2-upper D12S1485 CH2-lower/CH2-upper G00-595-295 D12S1485G00-595-416 CH5-lower/CH5-upper D12S1486 CH5-lower/CH5-upper G00-595-298D12S1486 G00-595-417 CH8-lower/CH8-upper D12S1487 CH8-lower/CH8-upperG00-595-301 D12S1487 G00-595-418 CH9-lower/CH9-upper D12S1488CH9-lower/CH9-upper G00-595-304 D12S1488 G00-595-419 EH2-lower/EH2-upperD12S1489 EH2-lower/EH2-upper G00-595-307 D12S1489 G00-595-420EH3-lower/EH3-upper D12S1490 EH3-lower/EH3-upper G00-595-310 D12S1490G00-595-421 EH4-lower/EH4-upper D12S1491 EH4-lower/EH4-upper G00-595-313D12S1491 G00-595-422 RM13-lower/RM13-upper D12S1492RM13-lower/RM13-upper G00-595-316 D12S1492 G00-595-423RM14-lower/RM14-upper D12S1493 RM14-lower/RM14-upper G00-595-319D12S1493 G00-595-424 RM16-lower/RM16-upper D12S1494RM16-lower/RM16-upper G00-595-322 D12S1494 G00-595-425RM25-lower/RM25-upper D12S1507 RM26-lower/RM26-upper G00-595-325D12S1495 G00-595-426 RM26-lower/RM26-upper D12S1495RM-29-lower/RM29-upper G00-595-328 D12S1496 G00-595-427RM31-lower/RM31-upper D12S1497 RM31-lower/RM31-upper G00-595-331D12S1497 G00-595-428 RM33-lower/RM33-upper D12S1498RM33-lower/RM33-upper G00-595-334 D12S1498 G00-595-429RM34-lower/RM34-upper D12S1499 RM34-lower/RM34-upper G00-595-337D12S1499 G00-595-430 RM36-lower/RM36-upper D12S1500RM36-lower/RM36-upper G00-595-340 D12S1500 G00-595-431RM46-lower/RM46-upper D12S1501 RM46-lower/RM46-upper G00-595-343D12S1501 G00-595-432 RM48-lower/RM48-upper D12S1502RM48-lower/RM48-upper G00-595-346 D12S1502 G00-595-433RM51-lower/RM51-upper D12S1503 RM51-lower/RM51-upper G00-595-349D12S1503 G00-595-434 RM53-lower/RM53-upper D12S1504RM53-lower/RM53-upper G00-595-352 D12S1504 G00-595-435RM60-lower/RM60-upper D12S1505 RM60-lower/RM60-upper G00-595-355D12S1505 G00-595-436 RM69-lower/RM69-upper D12S1506RM69-lower/RM69-upper G00-595-358 D12S1506 G00-595-437RM72-lower/RM72-upper D12S1508 RM25-lower/RM25-upper G00-595-361D12S1507 G00-595-438 RM76-lower/RM76-upper D12S1509RM72-lower/RM72-upper G00-595-364 D12S1508 G00-595-439RM85-lower/RM85-upper D12S1510 RM76-lower/RM76-upper G00-595-367D12S1509 G00-595-440 RM86-lower/RM86-upper D12S1511RM85-lower/RM85-upper G00-595-370 D12S1510 G00-595-441RM90-lower/RM90-upper D12S1512 RM86-lower/RM86-upper G00-595-373D12S1511 G00-595-442 RM93-lower/RM93-upper D12S1513RM90-lower/RM90-upper G00-595-376 D12S1512 G00-595-443RM98-lower/RM98-upper D12S1514 RM93-lower/RM93-upper G00-595-379D12S1513 G00-595-444 RM99-lower/RM99-upper D12S1515RM98-lower/RM98-upper G00-595-382 D12S1514 G00-595-445RM-29-lower/RM29-upper D12S1496 RM99-lower/RM99-upper G00-595-385D12S1515 G00-595-446 RM103-lower/RM103-upper D12S1516RM103-lower/RM103-upper G00-595-388 D12S1516 G00-595-447RM108-lower/RM108-upper D12S1517 RM108-lower/RM108-upper G00-595-391D12S1517 G00-595-448 RM110-lower/RM110-upper D12S1518RM110-lower/RM110-upper G00-595-394 D12S1518 G00-595-449RM111-lower/RM111-upper D12S1519 RM111-lower/RM111-upper G00-595-397D12S1519 G00-595-450 RM121-lower/RM121-upper D12S1520RM121-lower/RM121-upper G00-595-400 D12S1520 G00-595-451RM130-lower/RM130-upper D12S1521 RM130-lower/RM130-upper G00-595-403D12S1521 G00-595-452 RM131-lower/RM131-upper D12S1522RM131-lower/RM131-upper G00-595-406 D12S1522 G00-595-453RM132-lower/RM132-upper D12S1523 RM132-lower/RM132-upper G00-595-409D12S1523 G00-595-454 RM133-lower/RM133-upper D12S1524RM133-lower/RM133-upper G00-595-412 D12S1524 G00-595-455

TABLE 4 FISH mapping of chromosome 12 breakpoints in primary benignsolid tumors to a subregion of MAR Fraction of tumors with breakpointsBreakpoint within main breakpoint Tumor type with MAR cluster region*Lipoma {fraction (6/6)} {fraction (6/6)} Pleomorphic salivary {fraction(7/7)} {fraction (5/7)} gland adenoma Uterine leiomyoma ⅞ ⅞ Hamartoma ofthe breast {fraction (1/1)} {fraction (1/1)} Fibroadenoma of the breast{fraction (1/1)} {fraction (1/1)} Hamartoma of the lung {fraction (8/9)}{fraction (8/9)} Angiomyxoma {fraction (1/1)} {fraction (1/1)} *Tumorsamples were collected and analyzed at the histopathology andcytogenetics facilities of the University of Bremen. A mixture of cosmidclones 27E12 and 142H1 was used as molecular probe in FISH analysis.

TABLE 5 Tumor/ Diversion Chrom. sources poly(A) primer- Clone Cell LinePoint Nuc. Sequences (10b) RACE cytogen. signal #A's set (SEQ ID NOS:103-159) exon/intron TAGGAAATGG| GTGAGTAATA 3 (DBD3) pCH108 Li-14/SV40after DBD3 TAGGAAATGG| AATACTCTGG 12|? 3q27 AGTAAA 26 ? pCH113Li-538/SV40 after DBD3 TAGGAAATGG| AATACTCTGG 12|? 3q27 AGTAAA 26 ?pCH234 #2528-90 after DBD3 TAGGAAATGG| AATACTCTGG 12|? (AGTAAA) 17pCH259 #2344-94 after DBD3 TAGGAAATGG| AATACTCTGG 12|? ? pCH260 #2344-94after DBD3 TAGGAAATGG| AATACTCTGG 12|? ? pCH148 #192 after DBD3TAGGAAATGG| CCTACTATTG 12|N.T.² 12 AATAAA 18 — pCH245 #568 92 after DBD3TAGGAAATGG| CCTACTATTG 12| AATAAA 17 pCH247 #568 92 after DBD3TAGGAAATGG| CCTACTATTG 12| AATAAA 53 pCH261 #2344-94 after DBD3TAGGAAATGG| CCTACTATTG 12| AATAAA 27 pCH212 #1321-89 after DBD3TAGGAAATGG| GGAAGTGTGA 12| AATAAA 26 pCH213 #1321-89 after DBD3TAGGAAATGG| GGAAGTGTGA 12| ? pCH228 #1321-89 after DBD3 TAGGAAATGG|GGAAGTGTGA 12| N.D. 13 pCH216 #1321-89 after DBD3 TAGGAAATGG| AACACAGGAC12| AATAAA 12 pCH229 #2100-89 after DBD3 TAGGAAATGG| AACACAGGAC 12|AATAAA 15 pCH232 #2100-89 after DBD3 TAGGAAATGG| AACACAGGAC 12| AATAAA25 pCH177 #2100-89 after DBD3 TAGGAAATGG| GTTTAATATT 12|3 2p21 AATAAA 29177 pCH191 #367 after DBD3 TAGGAAATGG| GTTTAATATT 12|3 AATAAA 49 (177)pCH210 #25 after DBD3 TAGGAAATGG| AAGAAGGCAG 12| AATAAA 22 pCH211#837-88 after DBD3 TAGGAAATGG| AAGAAGGCAG 12| AATAAA 22 pCH230 #837-88after DBD3 TAGGAAATGG| TAGGAGGTAG 12| AATAAA 27 pCH233 #2100-89 afterDBD3 TAGGAAATGG| TAGGAGGTAG 12| AATAAA 18 pCH111 #2100-89 after DBD3TAGGAAATGG| GGTGGCCATT 12|3 3q27 AATAAA 33 111-AB pCH112 Li-501/SV40after DBD3 TAGGAAATGG| GGTGGCCATT 12|3 3q27 AATAAA 33 111-AB pCH114Li-501/SV40 after DBD3 TAGGAAATGG| GACAATCTAC 12|12 3q27 (CATAAA) 24115-AB pCH115 Li-538/SV40 after DBD3 TAGGAAATGG| GACAATCTAC 12|12 3q27(CATAAA) 24 115-AB pCH147 Li-538/SV40 after DBD3 TAGGAAATGG| GTACAGAAGA12|9 12 AATAAA 23 147 pCH153 #192 after DBD3 TAGGAAATGG| GGGCATTCAG12|N.T.¹ 4p13 AATAAA 22 — pCH169 #203 after DBD3 TAGGAAATGG| GCAGTCTGTA12|? 8q22 AATAAA 26 169 pCH172 #294 after DBD3 TAGGAAATGG| TCTGTATCCT12|8q22-qter 8q22 AATAAA 24 172-AB pCH173 #294 after DBD3 TAGGAAATGG|ACACACTTCC 12|? 8q22 AATAAA 12 173-AB pCH174 #294 after DBD3 TAGGAAATGG|ATATTATCGA 12|8q22 8q22 AATAAA 15 174-AB pCH110 LM-30-1/SV40 after DBD3TAGGAAATGG| GAGGAGTTTT 12|12 14 AATAAA 17 110-AB pCH164 Myo168.1 afterDBD3 TAGGAAATGG| TAACACACGA 12|? x (GATAAA) 30 164-AB pCH165 Myo168.1after DBD3 TAGGAAATGG| TTACCTGCTC 12|14 x (AATAAC) 30 165-AB pCH168Myo196.4 after DBD3 TAGGAAATGG| GCTGGAGTGC 12|12 14 (CATAAA) 23 168pCH209 #837-88 after DBD3 TAGGAAATGG| GTCTCCTCCC 12| AATAAA 19 pCH217#2100-89 after DBD3 TAGGAAATGG| TTTNTCTCTT 12| ATTAAA 22 pCH219 #2100-89after DBD3 TAGGAAATGG| AGTCCAAGAA 12| (CATAAA) 17 pCH220 #3391-90 afterDBD3 TAGGAAATGG| CCAAACTCTG 12| AATAAA 28 pCH223 #CG592 after DBD3TAGGAAATGG| CTCCAGAAAC 12| AATAAA 24 pCH226 #3100-88 after DBD3TAGGAAATGG| AACTTCTTGA 12| ? ? pCH231 #2100-89 after DBD3 TAGGAAATGG|GAATGTCAGA 12| AATAAA 24 pCH246 #568-92 after DBD3 TAGGAAATGG|CCTGGAAGCT 12| AATAAA ? pCH248 #568-92 after DBD3 TAGGAAATGG| ATGGAGTCTC12| AATAAA 40 pCH249 #2617-93 after DBD3 TAGGAAATGG| ATGGAGTCTC 12|AATAAA 19 pCH251 #2617-93 after DBD3 TAGGAAATGG| ATGGAGTCTC 12| AATAAA19 pCH250 #2617-93 after DBD3 TAGGAAATGG| TTCCAGATAC 12| N.D. 13 pCH252#2617-93 after DBD3 TAGGAAATGG| TTCCAGATAC 12| N.D. 13 exon/intronGCCTGCTCAG|GTAAGACATA 4 (SPACER) pCH109 LM30.1/SV40 after spacerGCCTGCTCAG| GTCAATGTTG 12|12 14 AATAAA 17 109-AB pCH238 #2778-93 afterspacer GCCTGCTCAG| GTCAATGTTG 12|12 14 AATAAA 17 109-AB pCH244 #2162-91after spacer GCCTGCTCAG| GTCAATGTTG 12|12 14 AATAAA 17 109-AB pCH254#2776-93 after spacer GCCTGCTCAG| GTCAATGTTG 12|12 14 AATAAA 17 109-ABpCH203 #2528-90 after spacer GCCTGCTCAG| TCCTGGTACC 12|(NF1″) N.D. 19pCH199 #CG575 after spacer GCCTGCTCAG| TCCTGGTACC 12|(NF1″) 12 (AATAAA)15 pCH222 #CG575 after spacer GCCTGCTCAG| TCCTGGTACC 12|(NF1″) 12 N.D.16 pCH206 #CG575 after spacer GCCTGCTCAG| TCCTGGTACC 12|(NF1″) 12(AATAAA) 16 pCH175 #275 after spacer GCCTGCTCAG| TCTTTCAGAT 12|? 1AATAAA 15 175-AB pCH237 #2617-93 after spacer GCCTGCTCAG| TCTTTCAGAT12|? AATAAA 16 pCH207 #2540-87 after spacer GCCTGCTCAG| AATTACCTCT 12|12 AATAAA 18 pCH239 #2344-94 after spacer GCCTGCTCAG| AATTACCTCT 12|AATAAA 18 pCH116 Li-14/SV40 after spacer GCCTGCTCAG| GACTGACTCA 12|12 3(AATAGA) 17 116-AB pCH184 Myo163.1 after spacer GCCTGCTCAG| TATTCCTGAA12|12 14 AATAAA 19 184-AB pCH208 #2540-87 after spacer GCCTGCTCAG|GTCAATGTTG 12| 12 AATAAA 47 pCH224 #2540-87 after spacer GCCTGCTCAG|AATTACCTCT 12| 12 AATAAA 15 pCH201 #837-88 after spacer GCCTGCTCAG|AATTACCTCT 12| AATAAA 15 pCH236 #2162-91 after spacer GCCTGCTCAG|GCTTTTTCAA 12| AATAAA 28 pCH243 #2162-91 after spacer GCCTGCTCAG|GTTAAGAAAC 12| ? ? pCH227 #183-89 after spacer GCCTGCTCAG| GNCTGACTAC12| 12 (AATAGA) 14 3′-untranslated region (various positions) pCH193 #58within TATCCTTTCA| AAGTCAAGAG 12|8q22-qter 8q24 AATAAA 23 (195-AB)3′-UTR pCH194 #58 within TATCCTTTCA| AAGTCAAGAG 12|8q22-qter 8q24 AATAAA34 (195-AB) 3′-UTR pCH195 #192 within TATCCTTTCA| AAGTCAAGAG 12|8 12AATAAA 23 195-AB 3′-UTR pCH196 #192 within TATCCTTTCA| AAGTCAAGAG 12|812 AATAAA >17 195-AB 3′-UTR pCH189 Myo192.1 within TCTTTCCACT| delCCCTGT 12|12 — — 3′-UTR ATACCACTTA| TTTTAAAACA 12|N.T.¹ 2/3 AATAAA 23 —pCH117 Ad-312/SV40 within TTGCCATGGT| AATCTGAAAT 12|1p22 1p22 ? 117-AB3′-UTR pCH253 #2617-93 within CACTTTCATC| ATATGGCAAG 12| N.D. 3′-UTRpCH264 #568-92 within ATAAGGACTA| TCAGGCATCA 12| (AGTAAA) 19 3′-UTRpCH270 #2528-90 within NCTTGTNAGC| TAGAGATTAG 12| N.D. 4 3′-UTR N.T.:NOT TESTABLE N.T.¹: LENGTH OF ECTOPIC SEQUENCE DOES NOT ALLOWDEVELOPMENT OF PRIMER-SET N.T.²: ECTOPIC SEQUENCE IS MAINLY COMPOSED OFREPETIVE SEQUENCES N.D.: NOT DETECTED

Legends to the Figures

FIG. 1

Long range physical map of a 6 Mb region on the long arm of humanchromosome 12 deduced from a YAC contig consisting of 75 overlappingCEPH YAC clones and spanning the chromosome 12q breakpoints as presentin a variety of benign solid tumors. The long range physical map of thecomposite genomic DNA covered by the YAC inserts is represented by ablack solid line with the relative positions of the various restrictionsites of rare cutting enzymes indicated. DNA regions in which additionalcutting sites of a particular restriction enzyme might be found areindicated by arrows. Polymorphic restriction endonuclease sites aremarked with asterisks. DNA markers isolated and defined by others aredepicted in green. DNA markers obtained by us are shown in boxes and arelabelled by an acronym (see also Table I and II). The relative positionsof these DNA markers in the long range physical map are indicated andthose corresponding to particular YAC ends are linked to these by adotted line. Some of the DNA markers have been assigned to a DNAinterval and this is indicated by arrows. For DNA markers in white boxesSTSs have been developed and primer sets are given in Table II. Forthose in yellow boxes, no primer sets were developed. The DNA intervalscontaining RAP1B, EST01096, or IFNG are indicated. Where applicable, Dnumber assignments are indicated. Below the long range physical map, thesizes and relative positions of the overlapping YAC clones fittingwithin the consensus long range restriction map are given as solid bluelines. DNA regions of YAC inserts not fitting within the consensus longrange restriction map are represented by dotted blue lines. CEPHmicrotiter plate addresses of the YAC clones are listed. The orientationof the YAC contig on chromosome 12 is given. The relative positions ofULCR12 and MAR are indicated by red solid lines labelled by thecorresponding acronyms. Accession numbers of STSs not listed in Table I:CH9 (#U27142); RM1 (#U29049); RM110 (#U29022); RM111 (#U29023); RM130(#U27139); RM131 (#U29001); RM132 (#U27138); RM133 (#U27137).Restriction sites: B: BssHII; K: KspI (=SacII); M: MluI; N: NotI; P:PvuI; Sf: SfiI.

FIG. 2

Contig of overlapping cosmids, long range restriction and STS mapspanning a segment of MAR of about 445 kb. Contig elements are numberedand defined in the list below. LL12NC01-derived cosmid clones are namedafter their microtiter plate addresses. GenBank accession numbers (#) ofthe various STSs are listed below. STSs are given in abbreviated form;e.g. RM33 instead of STS 12-RM33. A 40 kb gap between STSs “K” and “O”in the cosmid contig was covered by λ clones (clones 38 and 40) and PCRproducts (clones 37 and 39). The orientation of the contig on the longarm of chromosome 12 is given as well as the order of 37 STSs (indicatedin boxes or labelled with encircled capital letters). The slanted linesand arrows around some of the STS symbols at the top of the figure markthe region to which the particular STS has been assigned. It should benoted that the cosmid contig is not scaled; black squares indicate STSsof cosmid ends whereas the presence of STSs corresponding to internalcosmid sequences are represented by dots. Long range restriction map:Bs: BssHII; K: KspI (=SacII); M: MluI; N: NotI; P: PvuI; Sf: SfiI. Atthe bottom of the figure, detailed restriction maps are shown of thoseregions containing exons (boxes below) of the HMGI-C gene. Noncodingsequences are represented by open boxes and coding sequences by blackboxes. Estimated sizes (kb) of introns are as indicated. The relativepositions of the translation initiation (ATG) and stop (TAG) codons inthe HMGI-C gene as well as the putative poly-adenylation signal areindicated by arrows. Detailed restriction map: B: BamHI; E: EcoRI; H:HindIII. MAR: Multiple Aberration Region; DBD: DNA Binding Domain.

 1 = 140A3 30 = 46G3  2 = 202A1 31 = 59A1  3 = 78F11 32 = 101D8  4 =80C9 33 = 175C7  5 = 109B12 34 = 185H2  6 = 148C12 35 = 189C2  7 = 14H636 = 154B12  8 = 51F8 37 = pRM150  9 = 57C3 38 = pRM144 10 = 86A10 39 =PKXL 11 = 142G8 40 = pRM147 12 = 154A10 41 = 128A2 13 = 163D1 42 = 142H114 = 42H7 43 = 204A10 15 = 113A5 44 = 145E1 16 = 191H5 45 = 245E8 17 =248E4 46 = 154F9 18 = 33H7 47 = 62D8 19 = 50D7 48 = 104A4 20 = 68B12 49= 184A9 21 = 124D8 50 = 56C2 22 = 128A7 51 = 65E6 23 = 129F9 52 = 196E124 = 181C1 53 = 215A8 25 = 238E1 54 = 147G8 26 = 69B1 55 = 211A9 27 =260C7 56 = 22D8 28 = 156A4 57 = 116B7 29 = 27E12 58 = 144D12 A = STS12-EM12 (#U27145) I = STS 12-CH12 (#U27153) Q = STS 12-RM120 (#U27161) B= STS 12-EM30 (#U27146) J = STS 12-EM10 (#U27154) R = STS 12-RM118(#U27162) C = STS 12-EM14 (#U27147) K = STS 12-EM37 (#U27155) S = STS12-RM119 (#U27163) D = STS 12-EM31 (#U27148) L = STS 12-RM146 (#U27156)T = STS 12-EM2 (#U27164) E = STS 12-CH11 (#U27149) M = STS 12-RM145(#U27157) U = STS 12-EM4 (#U27165) F = STS 12-EM18 (#U27150) N = STS12-RM151 (#U27158) V = STS 12-EM3 (#U27166) G = STS 12-EM11 (#U27151) O= STS 12-EM16 (#U27159) W = STS 12-EM15 (#U27167) H = STS 12-CH10(#U27152) P = STS 12-EM1 (#U27160) X = STS 12-EM17 (#U27168) STS 12-CH5(#U27136) STS 12-CH9 (#U27142) STS 12-RM33 (#U27131) STS 12-RM53(#U27134) STS 12-RM76 (#U27132) STS 12-RM86 (#U27133) STS 12-RM98(#U26647) STS 12-RM99 (#U27130) STS 12-RM103 (#U26689) STS 12-RM130(#U27139) STS 12-RM132 (#U27138) STS 12-RM133 (#U27137) STS 12-RM151(#U27158)

FIG. 3

Schematic representation of FISH mapping data obtained for tumor celllines with chromosome 12q13-q15 aberrations, including 8 lipoma, 10uterine leiomyoma, and 8 pleomorphic salivary gland adenoma cell linesin consecutive experiments following our earlier FISH studies. Probesused included phage clones pRM144 (corresponding STSs: RM86 and RM130)and pRM147 (RM151), and cosmid clones 7D3 or 152F2 (RM103), 154F9 (CH9),27E12 (EM11), 211A9 (RM33), 245E8 (RM53), 185H2 (RM76), 202A1 (RM98),142H1 (RM99), 154B12 (RM132), and 124D8 (RM133). The DNA intervalbetween RM33 and RM98 is estimated to be about 445 kb. Dots indicateconclusive FISH experiments that were performed on metaphase chromosomesof a particular cell line using as molecular probe, a clone containingthe STS given in the box above. Solid lines indicate DNA intervals towhich a breakpoint of a particular cell line was concluded to bemapping. Open triangles indicate deletions observed during FISHanalysis. Open circles indicate results of FISH experiments on metaphasechromosomes of Li-501/SV40 cells with hybridization signals on acytogenetically normal chromosome 3. The positions of chromosome 12breakpoints of tumor cell lines mapping outside MAR are indicated byarrows. The molecularly cloned breakpoints of LM-30.1/SV40 andLM-608/SV40 are indicated by asterisks. Breakpoints in various uterineleiomyoma cell lines splitting cosmid 27E12 (EM1l) are indicated by“across”.

FIG. 4

3'-RACE product (SEQ ID Nos. 160-161) comprising the junction betweenpart of the HMGI-C gene and part of the LPP gene. The primers used andthe junction are indicated. The cDNA synthesis was internally primed andnot on the true poly(A) tail.

FIG. 5

Partial cDNA sequence of the LPP gene (SEQ ID NO:162).

FIG. 6

Amino acid sequence of the LPP gene (SEQ ID NO: 163). LIM domains areboxed. The breaking point is indicated with an arrow.

FIG. 7

Nucleotide sequence of HMGI-C (U28749) (SEQ ID NO:164 ). Thetranscription start site indicated as proposed by Manfioletti et al.[67] was arbitrarily chosen as a start site. The sequence contains thecomplete coding sequence.

FIG. 8

Gel of PCR products obtained as described in Example 5.

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Annex 1

Genes, Chromosome & Cancer 12:296-303 (1995)

Molecular Characterization of MAR, a Multiple Aberration Region on HumanChromosome Segment 12q13-q15 Implicated in Various Solid Tumors

Wim J. M. Van de Ven, Eric F. P. X. Schoen-akers, Sylke Wanschura, BerndRazmierczak, Patrick F. J. Kools, Jan M. W. Geurts, Sabine Bartnitzke,Herman Van den Berghe, and Jörn Bullerdiek

Center for Human Genetics, University of Leuven, Belgium (W. J. M. V. D.V., E. F. P. M. S., P. F. J. K., J. W. M. G., H. V. D. B.);

Center for Human Genetics, University of Bremen, Germany (S. W., B. K.,S. B., J. B.).

Chromosome arm 12q breakpoints in seven cell lines derived from primarypleomorphic salivary gland adenomas were mapped by FISH analysisrelative to nine DNA probes. These probes all reside in a 2.8 Mb genomicDNA region of chromosome segment 12q13-q15 and correspond to previouslypublished sequence-tagged sites (STS). Their relative positions wereestablished on the basis of YAC cloning and long range physical and STScontent mapping. The 12q breakpoints of five of the cell lines werefound to be mapping within three different subregions of the 445 kb DNAinterval that was recently defined as the uterine leiomyoma clusterregion of chromosome 12 breakpoints (ULCR12) between STS RM33 and RM98.All seven breakpoints appeared to map within the 1.7 Mb DNA regionbetween STS RM36 and RM103. Furthermore, the chromosome 12 breakpointsof three primary pleomorphic salivary gland adenomas were also found tobe mapping between RM36 and RM103. Finally, FISH analysis of two lipomacell lines with 12q13-q15 aberrations pinpointed the breakpoints ofthese to relatively small and adjacent DNA segments which, as well asthose of two primary lipomas, appeared to be located also between RM36and RM103. We conclude from the observed clustering of the 12qbreakpoints of the three distinct solid tumor types that the 1.7 Mb DNAregion of the long arm of chromosome 12 between RM36 and RM103 is amultiple aberration region which we designate MAR. Genes ChromosomCancer 12:296-303 (1995). © 1995 Wiley-Liss, Inc.

Introduction

Chromosome translocations involving region q13-15 of chromosome 12 havebeen observed in a wide variety of solid tumors (Mitelman, 1991). Insubgroups of cytogenetically abnormal uterine leiomyomas (Nilbert andHeim, 1990; Pandis et al., 1991), pleomorphic salivary gland adenomas(Sandros et al., 1990; Bullerdiek et al., 1993), and benign adiposetissue tumors (Sreekantaiah et al., 1991), 12q13-q15 aberrations arefrequently observed. In a recent study (Schoenmakers et al., 1994b), weidentified and molecularly characterized ULCR12, the uterine leiomyomacluster region of chromosome 12 breakpoints. In the present study, wefocus on the chromosome arm 12q breakpoints in pleomorphic adenoma ofthe salivary glands, a benign epithelial tumor originating from themajor or minor salivary glands. It is the most common type of salivarygland tumor and accounts for almost 50% of all neoplasms in theseorgans. About 85% of the tumors are found in the parotid gland, 10% inthe minor salivary glands, and 5% in the submandibular gland (Seifert etal., 1986). Although many of these adenomas appear to have a normalkaryotype, cytogenetic studies have also revealed recurrent specificchromosome anomalies (Sandros et al., 1990; Bullerdiek et al., 1993).Besides chromosome 8 aberrations, often translocations with a breakpointin 8q12 with, as the most common aberration, a t(3;8) (p21;q12),aberrations of chromosome 12, usually translocations involving12q13-q15, are also frequent. Non-recurrent clonal abnormalities havealso been described. The frequent involvement of region 12q13-q15 indistinct solid tumor types suggests that this chromosomal region harborsgene(s) that might be implicated in the evolution of these tumors.Molecular cloning of the chromosome 12 breakpoints of these tumors andcharacterization of the junction fragments may therefore lead to theidentification of such gene(s).

On the basis of fluorescence in situ hybridization (FISH) data, we havepreviously reported that the chromosome 12 breakpoints in a number ofcell lines derived from primary pleomorphic salivary gland adenomas(Kazmierczak et al., 1990; Schoenmakers et al., 1994a), are located onthe long arm of chromosome 12 in the interval between loci D12S19 andD12S8 (Schoenmakers et al., 1994a). This DNA interval has been estimatedto be about 7 cM (Keats et al., 1989; Craig et al., 1993). The intervalcontaining the chromosome 12 breakpoints of these tumor cells wasnarrowed further by showing that all breakpoints mapped distally to theCHOP gene, which is directly affected by the characteristic t(12;16)translocation in myxoid liposarcomas (Aman et al., 1992; Crozat et al.,1993; Rabbitts et al., 1993) and is located between D12S19 and D12S8. Inmore recent studies (Kools et al., 1995), the chromosome 12 breakpointof pleomorphic salivary gland adenoma cell line Ad-312/SV40 waspinpointed to a DNA region between sequence-tagged sites (STSs) RM110and RM111, which is less than 165 kb in size. FISH evaluation of thechromosome 12 breakpoints of the other pleomorphic salivary glandadenoma cell lines indicated that they must be located proximally to theone in Ad-312/SV40, at a distance of more than 800 kb (Kools et al.,1995). These results pointed towards a possible dispersion of thechromosome 12 breakpoints over a relatively large genomic region on thelong arm of chromosome 12.

Here, we report physical mapping of the chromosome 12 breakpoints inpleomorphic salivary gland adenoma cells from primary tumors as well asestablished tumor cell lines. The karyotypic anomalies observed in thecells were all different but always involved region q13-q15 ofchromosome 12. Using DNA probes between D12S8 and CHOP, whichcorresponded to sequence-tagged sites (STSs) of a long-range physicalmap of a 6 Mb DNA region and were obtained during chromosome walkingexperiments, we performed FISH experiments and defined more precisely amajor chromosome 12 breakpoint cluster region of pleomorphic salivarygland adenoma. This breakpoint cluster region appeared to overlap withULCR12. Furthermore, we tested whether 12q13-q15 breakpoints of lipomasmight also map within the same region as those of pleomorphic salivarygland adenoma and uterine leiomyoma.

Materials and Methods Primary Solid Tumors and Derivative Cell Lines.

Primary solid tumors including pleomorphic salivary gland adenomas,lipomas, and uterine leiomyomas were obtained from the UniversityClinics in Leuven, Belgium (Dr. I. De Wever); in Bremen, Germany (Dr. R.Chille); in Krefeld, Germany (Dr. J. Haubrich); and from the Instituteof Pathology in Göteborg, Sweden (Dr. G. Stenman). For cell culturingand subsequent FISH analysis, tumor samples were finely minced, treatedfor 4-6 hours with 0.8% collagenase (Boehringer, Mannheim, FRG), andprocessed further for FISH analysis according to routine procedures.

Human tumor cell lines used in this study included the previouslydescribed pleomorphic salivary gland adenoma cell lines Ad-211/SV40,Ad-248/SV40, Ad-263/SV40, Ad-295/SV40, Ad-302/SV40, AD-366/SV40, andAd-386/SV40 (Kazmierczak et al., 1990; Schoenmakers et al., 1994a) andthe lipoma cell lines Li-14/SV40 (Schoenmakers et al., 1994a) andrecently developed Li-538/SV40. Chromosome 12 aberrations found in thesecell lines are listed in Table 1. Cells were propagated in TC199 culturemedium with Earle's salts supplemented with 20% fetal bovine serum.

TABLE 1 Chromosome 12 Abberrations in Primary Human Solid Tumors andCell Lines* Aberration Cell lines Ad-211/SV40 t(8; 12)(q21; q13-q15)Ad-248/SV40 ins(12; 6)(q15; q16q21) Ad-263/SV40 inv(12)(q15q24.1)Ad-295/SV40 t(8; 12; 18)(p12; q14; p11.2) Ad-302/SV40 t(7; 12)(q31; q14)Ad-366/SV40 inv(12)(p13q15) Ad-386/SV40 t(12; 14)(q13-q15; q13-q15)Li-14/SV40 t(3; 12)(q28; q13) Li-538/SV40 t(3; 12)(q27; q14) LM-5.1/SV40t(12; 15)(q15; q24) LM-30.1/SV40 t(12; 14)(q15; q24) LM-65/SV40 t(12;14)(q15; q24) LM-67/SV40 t(12; 14)(q13-q15; q24) LM-100/SV40 t(12;14)(q15; q24) LM-605/SV40 ins(12; 11)(q14; q21qter) LM-608/SV40 t(12;14)(q15; q24) LM-609/SV40 t(12; 14)(q15; q24) Primary tumors Ad-386t(12; 14)(q15; q11.2) Ad-396 t(3; 12) Ad-400 t(12; 16) Li-166 t(12; 12)Li-167 t(3; 12)(q28; q14-q15) LM-163.1 t(12; 14)(q14; q24) LM-163.2t(12; 14)(q14-q24) LM-1683 t(X; 12)(q22; q15) LM-192 t(2; 3; 12)(q35;p21; q14) LM-196.4 t(12; 14)(q14; q24) *Ad, pleomorphic salivary glandadenoma; Li, lipoma; uterine leiomyoma.

DNA probes.

In the context of a human genome project focusing on the long arm ofchromosome 12, we isolated cosmid clones cRM33, cRM36, cRM51, cRM69,cRM72, cRM76, cRM98, cRM103, and cRM133, from chromosome 12-specificarrayed cosmid library LLNL12NC01 (Montgomery et al., 1993). Furtherdetails of these cosmid clones have been reported at the SecondInternational Chromosome 12 Workshop (1994) and will be describedelsewhere (Kucherlapati et al., 1994). Briefly, initial screenings wereperformed using a PCR-based screening strategy (Green and Olson, 1990),followed by filter hybridization analysis as the final screening step,as previously described (Schoenmakers et al., 1994b). The cosmid cloneswere isolated using STSs derived from YAC clones. STSs were obtainedupon rescue of YAC insert-ends using a methodology involvingvectorette-PCR followed by direct solid phase fluorescent sequencing ofthe PCR products (Geurts et al., 1994) or from inter-Alu PCR (Nelson etal., 1989). Cosmid clones were grown and handled according to standardprocedures (Sambrook et al., 1989).

Cosmid clone cPK12qter, which maps to the telomeric region of the longarm of chromosome 12 (Kools et al., 1995) was used as a referencemarker.

Chromosome Preparations and Fluorescence In Situ Hybridization.

Metaphase spreads of the pleomorphic salivary gland adenoma cell linesor normal human lymphocytes were prepared as described before(Schoenmakers et al., 1993). To unambiguously establish the identity ofchromosomes in the FISH experiments, FISH analysis was performed afterGTG-banding of the same metaphase spreads. GTG-banding was performedessentially as described by Smit et al. (1990). In situ hybridizationswere carried out according to a protocol described by Kievits et al.(1990) with some minor modifications (Kools et al., 1994; Schoenmakerset al., 1994b). Cosmid and YAC DNA was labelled with biotin-11-dUTP(Boehringer Mannheim) or biotin-14-dATP (BRL, Gaithersburg) as describedbefore (Schoenmakers et al., 1994b). Specimens were analyzed on a ZeissAxiophot fluorescence microscope using a FITC filter (Zeiss). Resultswere recorded on Scotch (3M) 640 asa film.

Results

FISH Mapping of 12q Breakpoints in Cell Lines of Pleomorphic SalivaryGland Adenoma.

In previous studies (Schoenmakers et al., 1994a), we mapped thechromosome 12 breakpoints in a number of pleomorphic adenomas of thesalivary glands relative to various DNA markers and established thatthese were all located proximally to locus D12S8 and distal to the CHOPgene. This region is somewhat smaller than the 7 cM region encompassedby linkage loci D12S8 and D12S19 (Keats et al., 1989). Using YACcloning, a long range physical/STS map has been constructed coveringmost of that 7 cM region, as recently reported (Kucherlapati et al.,1994). Furthermore, numerous genomic clones (cosmid clones) have beenisolated and their relative positions within this map established(Kucherlapati et al., 1994). Nine of these cosmids, including cRM33,cRM36, cRM51, cRM69, cRM72, cRM76, cRM98, cRM103, and cRM133, were usedin FISH studies to establish the positions of the chromosome 12breakpoints of the seven cell lines derived from pleomorphic adenomas ofthe salivary glands (Table 1). The relative mapping order of these ninecosmid clones, which cover a genomic region on the long arm ofchromosome 12 of about 2.8 Mb, is indicated in FIG. 1 and the results ofFISH studies with the various cosmid probes are schematically summarizedin the same figure. As an illustration, FISH results obtained withmetaphase cells of cell line Ad-295/SV40 using cRM76 and cRM103 asprobes are shown in FIG. 2. It should be noted that for theidentification of chromosomes, pre-FISH GTG-banding was used routinely.On the basis of such banding, hybridization signals could be assignedconclusively to chromosomes of known identity; this was of majorimportance for cases with cross- or background hybridization signals, asthese were occasionally observed. When GTG-banding in combination withFISH analysis provided inconclusive results, either because of weakhybridization signals or rather vague banding, FISH experiments wereperformed with cosmid clone cPK12qter (Kools et al., 1995) as areference probe.

FISH analysis of metaphase chromosomes of each of the seven pleomorphicsalivary gland adenoma cell lines with cosmid cRM103 revealed that thiscosmid mapped distal to the chromosome 12 breakpoints of all seven celllines studied here. Metaphase chromosomes of six of the seven cell lineswere also tested with probe cRM69 and, in two cases, with cRM51. Theresults of the latter experiments were always consistent with thoseobtained with cRM103. Similar FISH analysis with cRM36 as probeindicated that this probe mapped proximal to all the breakpoints. Theseresults were always consistent with those obtained for five of the sevencell lines in experiments using cRM72. Altogether, the results of ourFISH studies indicated that the chromosome 12 breakpoints of all sevencell lines map between cRM36 and cRM103, which spans a genomic region ofabout 1.7 Mb.

Fine Mapping of 12q Breakpoints in Cell Lines Derived from PleomorphicAdenomas of the Salivary Glands.

For subsequent fine mapping of the chromosome 12 breakpoints of theseven pleomorphic salivary gland adenoma cell lines, additional FISHstudies were performed, as schematically summarized in FIG. 1. Thebreakpoints of cell lines Ad-211/SV40, Ad-295/SV40, and Ad-366/SV40appeared to be located in the DNA region between cRM76 and cRM133, whichwas estimated to be about 75 kb. The breakpoints of the four other celllines were found in different areas of the 1.7 Mb region between cRM36and cRM103. That of cell line Ad-248/SV40 in a DNA segment of about 270kb between cRM33 and cRM76, that of Ad-263/SV40 in a DNA segment ofabout 1 Mb between cRM98 and cRM103, that of Ad-302/SV40 in a DNAsegment of about 240 kb between cRM33 and cRM36, and that of Ad-386/SV40in a DNA segement of about 100 kb between cRM98 and cRM133. Inconclusion, these results indicated that the chromosome 12 breakpointsof most (5 out of 7) of the cell lines are dispersed over the 445kb-genomic region on the long arm of chromosome 12 between cRM33 andcRM98. It is important to note already here that precisely this regionwas recently shown to contain the chromosome 12q breakpoints in celllines derived from primary uterine leiomyomas (see FIG. 3) and wastherefore designated ULCR12 (Schoenmakers et al., 1994b). As thissegment of the long arm of chromosome 12 is involved in at least twotypes of solid tumors (Schoenmakers et al., 1994b; this study) and, aswe will show below, also in a third solid tumor type, we will from nowon refer to the DNA interval between cRM36 and cRM103 as MAR (multipleaberration region).

FISH Mapping of 12q Breakpoints in Primary Pleomorphic Salivary GlandAdenomas.

Our FISH studies on metaphase chromosomes of pleomorphic adenomas of thesalivary glands presented so far were restricted to cell lines derivedfrom primary tumors. Although it is reasonable to assume that thechromosome 12 breakpoints in cell lines are similar if not identical tothe ones in the corresponding primary tumors, differences as a result ofthe establishment of cell lines or subsequent cell culturing cannotfully be excluded. Therefore, we have investigated whether thechromosome 12 breakpoints in three primary salivary gland adenomas weremapping to MAR as well. To test this possibility, a combination ofcosmid clones cRM33 and cRM103 were used as molecular probe. In allthree cases, this cosmid pool clearly spanned the chromosome 12breakpoints (data not shown), indicating that these breakpoints wereindeed localized within MAR. In a recent study (Wanschura et al.,submitted for publication), it was reported that the chromosome 12breakpoints of five primary uterine leiomyomas with 12q14-q15aberrations were all found to cluster within the 1.5 Mb DNA fragment(between cRM33 and cRM103), which is known to harbor the breakpoints ofvarious cell lines derived from primary uterine leiomyomas(schematically summarized in FIG. 3). Consistent with the results of thebreakpoint mapping studies using cell lines, the results with the twoprimary solid tumor types establish that the breakpoints of the primarytumor cells are located in MAR.

Chromosome Segment 12q13-q15 Breakpoints of Lipomas Mapping within MA.

To test the possibility that the chromosome 12 breakpoints of othersolid tumors with 12q13-q15 aberrations also mapped within MAR, westudied two lipomas cell lines by FISH analysis—Li-14/SV40 andLi-538/SV40. The chromosome 12 aberrations of these two lipoma celllines are given in Table 1. As molecular probes, cosmid clones cRM33,cRM53, cRM72, cRM76, cRM99, cRM103, and cRM133 were used. The breakpointof Li-14/SV40 was mapped to the 75 kb DNA interval between RM76 andRM133, and that of Li-538/SV40 to the 90 kbp interval between RM76 andRM99 (data not shown), as schematically illustrated in FIG. 3. SimilarFISH analysis of two primary lipomas using a mixture of cRM36 and cRM103as molecular probe resulted in a hybridization pattern indicating thatthe mixture of probes detected sequences on either side of thebreakpoints. These results are the first indications that also inlipoma, chromosome 12q13-q15 breakpoints occur that map within MAR. Morelipoma cases should be tested to allow proper interpretation of thisobservation.

Discussion

In this study, we have mapped the chromosome 12 breakpoints of threeprimary pleomorphic salivary gland adenomas as well as seven establishedcell lines derived from such tumors. All breakpoints appeared to belocated in a previously molecularly cloned and characterized chromosomeDNA segment on the long arm of chromosome 12, of about 1.7 Mb in size,with five of them clustering in a DNA interval of less than 500 kb. The1.7 Mb DNA region apparently contains a major breakpoint cluster regionfor this type of tumor. In a previous study, we have described thecharacterization of the chromosome 12 breakpoint of pleomorphic salivarygland adenoma cell line Ad-312/SV40 (Kools et al., 1995). The breakpointof this cell line is now known to map at a distance of more than 2 Mbdistally to this major breakpoint cluster region reported here. It ispossible that the Ad-312/SV40 breakpoint involves other pathogeneticallyrelevant genetic sequences than those affected by the clusteredbreakpoints. However, the possibility should not yet be excluded thatall the 12q13-q15 breakpoints in pleomorphic salivary gland adenomasmapped so far belong to the same category and are dispersed over arelatively large DNA region of this chromosome, reminiscent of the 11q13breakpoints in B-cell malignancies (Raynaud et al., 1993). More precisepinpointing of the various breakpoints could shed more light on thismatter.

Of importance is the observation that the DNA segment that harbors theclustered 12q breakpoints of pleomorphic salivary gland adenomas appearsto coincide with the DNA region that was recently defined as the uterineleiomyoma cluster region of chromosome 12 breakpoints, known as ULCR12(Schoenmakers et al., 1994b). Of further interest is the fact that thisregion of chromosome 12 also harbors breakpoints of primary lipomas andlipoma cell lines derived from primary tumors with 12q13-q15aberrations. Altogether, the results of all these studies now clearlydemonstrate that chromosome 12 breakpoints of three distinct solid tumortypes map to the same 1.7 Mb genomic region on the long arm ofchromosome 12, establishing this region to be a multiple aberrationregion. To reflect this characteristic, we have designated this DNAsegment MAR.

Genetic aberrations involving chromosomal region 12q13-q15 have beenimplicated by many cytogenetic studies in a variety of solid tumorsother than the three already mentioned. Involvement of 12q13-q15 hasalso been reported for endometrial polyps (Walter et al., 1989; Vanni etal., 1993), clear cell sarcomas characterized by recurrentt(12;22)(q13;q13) (Fletcher, 1992; Reeves et al., 1992; Rodriguez etal., 1992), a subgroup of rhabdomyosarcoma (Roberts et al., 1992) andhemangiopericytoma (Mandahl et al., 1993a), chondromatous tumors(Mandahl et al., 1989; Bridge et al., 1992; Hirabayashi et al., 1992;Mandahl et al., 1993b), and hamartoma of the lung (Dal Cin et al.,1993). Finally, several case reports of solid tumors with involvement ofchromosome region 12q13-q15 have been published—e.g., tumors of thebreast (Birdsal et al., 1992; Rohen et al., 1993), diffuse astrocytomas(Jenkins et al., 1989), and a giant-cell tumor of the bone (Noguera etal., 1989). On the basis of results of cytogenetic studies, nopredictions could be made about the relative distribution of thebreakpoints of these tumor types. In light of the results of the presentstudy, it.would be of interest to see whether the breakpoints of any ofthese solid tumors also map within or close to MAR. The various cosmidclones available now provide the means to test this readily.

The observation that 12q breakpoints of at least three different typesof solid tumors map to the same DNA region is intriguing as it could bepointing towards the possibility that the same genetic sequences in MARare pathogenetically relevant for tumor development in differenttissues. If so, it is tempting to speculate that the gene(s) affected bythe genetic aberrations might be involved in growth regulation. On theother hand, one cannot yet exclude the possibility that geneticsequences in MAR are not pathogenetically relevant, as the observedclustering of genetic aberrations in MAR could simply reflect geneticinstability of this region, which becomes apparent in various solidtumors. To obtain more insight in this matter, the genes residing in MARshould be identified and characterized, and this can be achieved byvarious approaches using several techniques (Parrish and Nelson, 1993).

Acknowledgments

The constructive support of managing director G. Everaerts is greatlyacknowledged. The authors would like to thank P. Dal Cin, J. Haubrich,R. Hille, G. Stenman, and I. De Wever for providing the solid tumorspecimens studied in the present report; C. Huysmans, E. Meyen, K.Meyer-Bolte, R. Mols, and M. Willems for excellent technical assistance;and M. Leys for artwork. This work was supported in part by the ECthrough Biomed 1 program “Molecular Cytogenetics of Solid Tumours”, the“Geconcerteerde Onderzoekacties 1992-1996”, the National Fund forScientific Research (NFWO; Kom op tegen Kanker), the “ASLK-programmavoor Kankeronderzoek”, the “Schwerpunktprogramm: Molekulare undKlassische Tumorcytogenetik” of the Deutsche Forschungsgemeinschaft, andthe Tönjes-Vagt Stiftung. This text presents results of the Belgianprogramme on Interuniversity Poles of attraction initiated by theBelgian State, Prime Minister's Office, Science Policy Programming. Thescientific responsibility is assumed by its authors. J. W. M. Geurts isan “Aspirant” of the National Fund for Scientific Research (NFWO; Kom optegen Kanker).

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Schoenmakers H F M, Kools P F J, Mols R, Kazmierczak B, Bartnitzke S,Bullerdiek J, Dal Cin P, De Jong P J, Van den Berghe H, Van de Ven W J M(1994a) Physical mapping of chromosome 12q breakpoints in lipoma,pleomorphic salivary gland adenoma, uterine leiomyoma, and myxoidliposarcoma. Genomics 20: 210-222.

Schoenmakers H F P M, Mols R, Wanschura S, Kools P F J, Geurts J M,Bartnitzke S, Bullerdiek J, Van den Berghe H, Van de Ven W J M (1994b)Identification, molecular cloning and characterization of the chromosome12 breakpoint cluster region of uterine leiomyomas. Genes ChromosomCancer 11: 106-118.

Second International Chromosome 12 Workshop, New Haven, Conn., USA, Jun.20-22, 1994.

Seifert G, Miehlke A, Haubrich J, Chilla R (1986): Diseases of thesalivary glands. Pathology. Diagnosis. Treatment. Facial nerve surgery.Translated by P. M. Stell. Thieme, Stuttgart, New York, pp 182-194.

Smit V T H B M, Wessels J W, Mollevanger P, Schrier P I, Raap A K,Beverstock G C, Cornelisse C J (1990) Combined GTG-banding andnonradioactive in situ hybridization improves characterization ofcomplex karyotypes. Cytogenet Cell Genet 54:20-23.

Sreekantaiah C, Leong S L P, Karakousis C P, McGee D L, Rappaport W D,Villar H V, Neal D, Fleming S, Wankel A, Herrington P N, Carmona R,Sandberg A A (1991) Cytogenetic profile of 109 lipomas. Cancer Res 51:422-433.

Vanni R, Dal Cin P, Marras S, Moerman P h, Andria M, Valdes E, DeprestJ, and Van den Berghe H (1993) Endometrial polyp: Another benign tumorcharacterized by 12q13-q15 changes. Cancer Genet Cytogenet 68:32-33.

Walter T A, Xuan Fan S, Medchill M T, Berger C S, Decker H-J H, SandbergA A (1989) Inv(12) (p11.2q13) in an endometrial polyp. Cancer GenetCytogenet 41: 99-103.

Wanschura S, Belge G, Stenman G, Kools P, Dal Chin P, Schoenmakers E,Huysmans C, Bartnizke S, Van de Ven W, and Bullerdiek J (submitted forpublication). Mapping of the translocation breakpoints of primarypleomorphic adenomas and lipomas within a common region of chromosome12.

Legends of Figures of Annex 1

FIG. 1. Schematic representation of FISH mapping data obtained for theseven pleomorphic salivary gland adenoma cell lines tested in thisstudy. Cosmid clones which were used as probes in the FISH mappingstudies map at sequence-tagged sites obtained from overlapping YACclones. They are named after the acronyms of the STSs, as shown in theboxes, and the relative order of these is as presented. The DNA intervalbetween RM69 and RM72 is estimated to be about 2.8 Mb. The solid linesindicate DNA intervals in which the breakpoints of the various celllines are located. The dots indicate FISH experiments that wereperformed on metaphase chromosomes of the various cell lines using acosmid clone corresponding to the STS indicated above these as molecularprobe. The relative positions of MAR and ULCR12 are indicated in thelower part of the figure. Ad, pleomorphic salivary gland adenoma; MAR,multiple aberration region; ULCR12, uterine leiomyoma cluster region ofchromosome 12 breakpoints.

FIG. 2. a: Partial karyotype of Ad-295/SV40 showing der(8), der(12),der(18) and the corresponding normal chromosomes. b: FISH analysis ofmetaphase chromosomes of Ad-295/SV40 cells using DNA of cosmid clonecRM76 as molecular probe. Hybridization signals on normal chromosome 12(arrow) and der(12) (arrowhead). c: GTG-banding pattern of metaphasechromosomes of Ad-295/SV40 shown in b. d: FISH analysis of metaphasechromosomes of Ad-295/SV40 cells using DNA of cosmid clone cRM103 asmolecular probe. Hybridization signals on normal chromosome 12 (arrow)and der(18) (arrowhead).

FIG. 3. Schematic representation of chromosome 12 breakpoint mappingdata obtained for primary pleomorphic salivary gland adenomas, uterineleiomyomas, and lipomas as well as cell lines derived from such solidtumors. Results are compared to data for primary uterine leiomyomas(Wanschura et al., sumitted for publication) and cell lines derived fromsuch tumors (Schoenmakers et al., 1994b). Cosmid clones which were usedas probes in the FISH mapping studies correspond to sequence-taggedsites obtained from overlapping YAC clones. Cosmid clones were namedafter the acronyms of the STSs, as shown in the boxes, and the relativeorder of these is as presented. The estimated sizes of DNA intervalsbetween STSs are indicated. A d, pleomorphic salivary gland adenoma; Li, lipoma; L M, uterine leiomyoma.

Annex 2

Lead Article

Identification of the Chromosome 12 Translocation Breakpoint Region of aPleomorphic Salivary Gland Adenoma with t(1;12)(p22;q15) as the SoleCytogenetic Abnormality

Patrick F. J. Kools, Sylke Wanschura, Eric F. P. X. Schoenmakers, Jan W.M. Geurts, Raf Mols, Bernd Kazmierczak, Jörn Bullerdiek, Herman Van denBerghe and Wim J. M. Van de Ven

ABSTRACT: Cell line Ad-312/SV40, which was derived from a primarypleomorphic salivary gland adenoma with t(1;12)(p22;q15), was used influorescence in situ hybridization (FISH) analysis to characterize itstranslocation breakpoint region on chromosome 12. Results of previousstudies have indicated that the chromosome 12 breakpoint in Ad-312/SV40is located proximally to locus D12S8 and distally to the CHOP gene. Wehere describe two partially overlapping yeast artificial chromosome(YAC) clones, Y4854 (500kbp) and Y9091 (460 kbp), which we isolated inthe context of a chromosome walking project with D12S8 and CHOP asstarting points. Subsequently, we have isolated cosmid clonescorresponding to various sequence-tagged sites (STSs) mapping within theinserts of these YAC clones. These included cRM51, cRM69, cRM8S, cRM90,cRM91, cRM110, and cRM111. We present a composite long-range restrictionmap encompassing the inserts of these two YAC clones and show by FISHanalysis, that both YACs span the chromosome 12 breakpoint as present inAd-312/SV40 cells. In FISH studies, cosmid clones cRM85, cRM90 andcRM111 appeared to map distally to the chromosome 12 breakpoint whereascosmid clones cRM51, cRM69, cRM91, and cRM110 were found to mapproximally to it. These results assign the chromosome 12 breakpoint inAd-312/SV40 to a DNA region of less than 165 kbp. FISH evaluation of thechromosome 12 breakpoints in five other pleomorphic salivary glandadenoma cell lines indicated that these are located proximally to theone in Ad-312/SV40, at a distance of more than 0.9 Mb from STS RM91.These results, while pinpointing a potentially critical region onchromosome 12, also provide evidence for the possible involvement ofchromosome 12q13-q15 sequences located elsewhere.

Introduction

Pleomorphic salivary gland adenoma constitutes a benign epithelial tumorthat originates from the major and minor salivary glands. It is the mostcommon type of salivary gland tumor and accounts for almost 50% of allneoplasms in these organs; 85% of the tumors are found in the parotidgland, 10% in the minor salivary glands, and 5% in the submandibulargland [1]. About 50% of these adenomas appear to have a normal karyotypebut cytogenetic studies have also revealed recurrent specific chromosomeanomalies [2, 3]. Frequently observed anomalies include aberrations ofchromosome 8, usually involving the 8q12-q13 region, with the mostcommon aberration a t(3;8) (p21;q12), and aberrations of chromosome 12,usually translocations involving region 12q13-q15. Non-recurrent clonalchromosome abnormalities have also been reported. The highly specificpattern of chromosome rearrangements with consistent breakpoints at8q12-q13 and 12q13-q15 suggests that these chromosomal regions harbourgenes that might be implicated in the development of these tumors.Molecular cloning of the chromosome breakpoints and characterization oftheir junction fragments may lead to the identification ofpathogenetically relevant genes. At present, no such molecular data haveyet been reported for these tumors.

On the basis of fluorescence in situ hybridization (FISH) data, thechromosome 12 breakpoints in six pleomorphic salivary gland adenoma celllines were recently shown to be mapping to region 12q13-q15, moreprecisely, to the genomic interval between loci D12S19 and D12S8 [4, 5].The sex-averaged genetic size of this genomic DNA interval was reportedat HGM10 to be 7 cM [6]. We also reported that the chromosome 12breakpoints in salivary gland adenomas map distally to the CHOP gene[5], which supports an earlier study indicating that the 12q13-q15translocation breakpoints in pleomorphic salivary gland adenomas aredifferent from that in myxoid liposarcoma [7]. Here, we report about thephysical mapping of the chromosome 12 breakpoint in pleomorphic salivarygland adenoma cell line Ad-312/SV40, which carries a t(1;12) (p22;q15)as the only cytogenetic abnormality.

Materials and Methods

Tumor Cell Lines.

Human tumor cell lines used in this study included the previouslydescribed pleomorphic salivary gland adenoma cell lines Ad-248/SV40,Ad-263/SV40, Ad-295/SV40, Ad-302/SV40, Ad-312/SV40, and Ad-366/SV40 [5,8]. Cells were cultivated in TC199 culture medium with Earle's saltssupplemented with 20% fetal bovine serum. Other cell lines used in thisstudy included somatic cell hybrid PK89-12, which contains chromosome 12as the sole human chromosome in a hamster genetic background [9], andsomatic cell hybrid LIS-3/SV40/A9-B4 [4]. The latter cell line wasobtained upon fusion of myxoid liposarcoma cell line LIS-3/SV40, whichcarries the specific t(12;16)(q13;p11.2), with mouse A9 cells. Thissomatic cell hybrid was previously shown to contain der(16) but neitherder(12) nor the normal chromosome 12 [4]. PK89-12 and LIS-3/SV40/A9-B4cells were grown in DME-F12 medium supplemented with 10% fetal bovineserum. Cell lines were analyzed by standard cytogenetic techniques atregular intervals.

Isolation of YAC and Cosmid Clones.

In the context of human genome mapping studies, which will be describedin detail elsewhere (Schoenmakers et al., in preparation), we isolatedYAC clones Y4854 and Y9091 from the first-generation CEPH YAC library[10), and cosmid clones cRM51, cRM69, cRM85, cRM90, cRM91, cRM103,cRM110, and cRM111 from the chromosome-12-specific, arrayed cosmidlibrary LLNLNCO1 [11]. YAC and cosmid clones were isolated as describedbefore [5]. Initial screenings of the YAC, as well as the cosmidlibrary, were performed using a screening strategy involving thepolymerase chain reaction (PCR) [12]. Filter hybridization analysis wasused as the final screening step, as previously described [5]. Cosmidclones were isolated using STSs and those corresponding to STSs withinthe inserts of YAC clones Y4854 and Y9091 are indicated in FIG. 1. STSswere obtained via rescue of YAC insert end-sequences using avectorette-PCR procedure [13] or Alu-PCR [14, 15]. PCR products weresequenced directly via solid-phase fluorescent sequencing. Cosmid cloneswere grown and handled according to standard procedures [16]. YAC cloneswere characterized by pulsed-field gel electrophoresis [17], restrictionmapping, and hybridization, as previously described [5].

Chromosome Preparations and Fluorescence in situ Hybridization.

Cells from the pleomorphic salivary gland adenoma tumor cell lines weretreated with Colcemid (0.04 μg/ml) for 30 min and then harvestedaccording to routine methods. Metaphase spreads of the tumor cells wereprepared as described before [4]. To establish the identity ofchromosomes in the FISH experiments, FISH analysis was performed afterG-banding of the same metaphase spreads. G-banding was performedessentially as described by Smit et al. [18]. In situ hybridizationswere carried out according to a protocol described by Kievits et al.[19] with some minor modifications [5, 20]. Cosmid and YAC DNA waslabelled with biotin-11-dUTP (Boehringer Mannheim) or biotin-14-dATP(BRL, Gaithersburg), as described earlier (5]. Chromosomes werecounterstained with propidium iodide and analyzed on a Zeiss Axiophotfluorescence microscope using a FITC filter (Zeiss). Results wererecorded on Scotch (3M) 640asa film.

Results

Isolation and Characterization of YAC Clones Spanning the Chromosome 12Breakpoint of Pleomorphic Salivary Gland Adenoma Cell Line Ad-312/SV40.

In previous studies [5], we mapped the chromosome 12 breakpoints of sixpleomorphic salivary gland adenoma cell lines proximally to locus D12S8and distally to CHOP. The DNA interval between these loci is somewhatsmaller than 7 cM (estimated distance between the loci D12S8 and D12519[6]) but still substantially large. To molecularly define thetranslocation breakpoint of Ad-312/SV40, we have performed human genomemapping studies on the DNA interval between locus D12S8 and the CHOPgene. In the process of directional chromosome walking starting fromD12S8 and the CHOP gene, we obtained overlapping YAC clones Y9091 andY4854. The DNA insert of Y9091 appeared to be 460 kbp and that of Y4854,500 kbp. Moreover, as we will demonstrate below, the DNA insert of eachYAC clone appeared to span the chromosome 12 breakpoint of Ad-312/SV40.A long-range restriction map of the inserts of these YAC clones was madeusing pulsed-field gel electrophoresis and hybridization analysis (FIG.1). On the basis of STS content mapping and Southern blot analysis, theinserts of YAC clones Y9091 and Y4854 appeared to overlap as indicatedin FIG. 1. The tested STSs correspond to end-sequences of otheroverlapping YAC clones not shown here or to sequences obtained viainter-Alu-PCR. Of these, RM90 and RM91 represent such end-clone STSs ofYAC Y9091, and RM48 and RM54 of Y4854, whereas RM110 and RM111 representSTSs derived from inter-Alu-PCR. For a number of STSs mapping within theinserts of YAC clones Y4854 and Y9091, corresponding cosmid clones wereisolated for use in FISH analysis, e.g., cRM51, cRM69, cRM85, cRM90,cRM91, cRM110, and cRM111.

The inserts of the two overlapping YAC clones are most likely notchimeric, as was deduced from the following observations. FISH analysisof metaphase chromosomes of normal human lymphocytes with Y4854 or Y9091DNA as molecular probe revealed hybridization signals only in chromosomeregion 12q13-q15. For Y9091, this was confirmed further by observationsmade in FISH studies in which cosmid clone cRM90 or cRM91 was used asprobe; the DNA insert of each of these two cosmids corresponds to thealternative end-sequences of YAC clone Y9091. Finally, the end-sequenceSTSs of Y9091 appeared to map to chromosome 12 and distally to the CHOPgene, as was established by PCR analysis on PK89-12 DNA, which containshuman chromosome 12 as the sole human chromosome in a hamster geneticbackground, and LIS-3/SV40/A9-B4 DNA, which was previously shown tocontain der(16), from the specific t(12;16) of myxoid liposarcoma, butneither der(12) nor the normal chromosome 12 [4]. From the chromosomewalking studies, we concluded that the overlapping inserts of the twoYAC clones represent a DNA region of about 640-kbp, which is located onchromosome 12q between D12S8 and CHOP. As the 640-kbp compositelong-range restriction map of the YAC contig was constructed with atleast double coverage of the entire region, it is not unreasonable toassume that the 640-kbp region is contiguous with the chromosomal DNA,although microdeletions can not be excluded at this point.

Chromosome walking was routinely evaluated by FISH mapping of YAC clonesand/or cosmid clones corresponding to YAC insert sequences. It should benoted that for the identification of chromosomes, G-banding was used inmost cases. On the basis of such G-banding, hybridization signals couldbe assigned conclusively to chromosomes of known identity; this was alsoof importance for the cases with cross- or background hybridizationsignals that were occasionally observed. G-banding prior to FISHanalysis resulted sometimes in rather weak hybridization signals orrather vague banding patterns. Therefore, we performed FISH experimentsin which the YAC and cosmid clones to be evaluated were used incombination with a reference probe. Cosmid clone cPK12qter, which wasserendipitously obtained during screening of a cosmid library, wasselected as reference marker. FISH analysis of metaphase chromosomes ofnormal lymphocytes (FIG. 2A) revealed that cPK12qter maps to thetelomeric region of the long arm of chromosome 12. To identifychromosome 12 in this experiment, centromere 12-specific probe pα12H8[21] was used. FISH analysis of metaphase chromosomes of Ad-312/SV40cells using YAC clone Y4854 (FIG. 2B) or Y9091 (FIG. 2C) in combinationwith reference probe cPK12qter revealed, in both cases, hybridizationsignals of the YAC insert on der(1) as well as der(12). We concludedfrom these results that the insert DNA of each YAC clone might span thechromosome 12 breakpoint in this cell line. It should be noted thatG-banding revealed a telomeric association involving the short arm ofchromosome 12 in FIG. 2C. The observation that YAC clone Y9091 spannedthe chromosome 12 breakpoint in Ad-312/SV40 was confirmed independentlyin FISH studies in which cosmid clone cRM90 or cRM91 was used asmolecular probe; they were shown to contain the alternativeend-sequences of the Y9091 insert. cRM90 appeared to map distally to thechromosome 12 breakpoint, whereas cRM91 was found to map proximally(data not shown). These results also established the chromosomalorientation of the YAC contig shown in FIG. 1. In summary, we concludedfrom these FISH studies that the chromosome 12 translocation breakpointin Ad-312/SV40 must be located in the DNA interval corresponding to theoverlapping sequences (about 300 kbp) of the two YAC clones.

Fine Mapping of the Chromosome 12 Translocation Breakpoint ofAd-312/Sv40.

In an approach to further narrow the chromosome 12 translocationbreakpoint region of Ad-312/SV40, cosmid clones with different mappingpositions within YAC clone Y9091 were isolated. These included cRM69,cRM85, cRM110, and cRM111. cRM69 and cRM85 were isolated on the basis ofSTS sequences of YAC clones not shown here. cRM110 and cRM111 wereobtained via inter-Alu-PCR. RM110 was shown by Southern blot analysis tohybridize to a terminal MluI fragment of Y9091 and not to the DNA insertof the overlapping YAC clone with RM69 as telomeric end-sequences. Thelocation of RM110 is as indicated in FIG. 1. RM111 was shown tohybridize to a BssHII, MluI, PvuI, and SfiI fragment of Y9091 and istherefore located in the PvuI-SfiI fragment of Y9091, to which STS RM48was also mapped (FIG. 1). FISH analysis of metaphase chromosomes ofAd-312/SV40 with cRM69 or cRM110as probe indicated that the DNA insertof these cosmids mapped proximally to the chromosome 12 translocationbreakpoint in this cell line, as illustrated, for cRM69 in FIG. 3A.Subsequent FISH analysis of Ad-312/SV40 with cRM85 or cRM111as proberevealed hybridization signals distally to the translocation breakpoint,as illustrated for cRM111 in FIG. 3B. The results with cRM85 and cRM111are in agreement with the observed breakpoint spanning by YAC cloneY4854 as cRM85 maps distally and cRM111 closely to STS RM48, which marksthe telomeric end of YAC clone Y4854. In conclusion, the chromosome 12translocation breakpoint in Ad-312/SV40 must be located in the DNAinterval between cRM110 and cRM111, as schematically summarized in FIG.4.

FISH Evaluation of Chromosome 12 Breakpoints in Other PleomorphicSalivary Gland Adenoma Cell Lines.

To determine the position of their chromosome 12 breakpoints relative tothat of Ad-312/SV40, five other pleomorphic salivary gland adenoma celllines were evaluated by FISH analysis, as summarized schematically inFIG. 4. These cell lines, which were developed from primary tumors [5,8], included Ad-248/SV40, Ad-263/SV40, Ad-295/SV40, Ad-302/SV40, andAd-366/SV40. The chromosome 12 aberrations of these cell lines arelisted in FIG. 4. FISH analysis of metaphase chromosomes of these celllines using cRM91 revealed that the chromosome 12 breakpoints of allthese cell lines mapped proximally to this cosmid clone (data notshown). Similar FISH analysis was also performed using a cosmid clonecorresponding to sequence-tagged site RM103 as a probe. RM103 was foundto map proximally to RM91 at a distance of about 0.9 Mbp. In all cases,cRM103 appeared to map distally to the chromosome 12 translocationbreakpoints, indicating that the chromosome 12 breakpoints in these fivepleomorphic salivary gland adenoma cell lines are located at arelatively large distance from that of Ad-312/SV40 cells.

Discussion

In the studies presented here, we have identified, molecularly cloned,and characterized a chromosome region on the long arm of chromosome 12in which the translocation breakpoint of pleomorphic salivary glandadenoma cell line Ad-312/SV40 appears to map. In previous studies [5],we already provided evidence that the chromosome 12 breakpoint of thiscell line was located between D12S8 and CHOP. Because the twobreakpoints spanning YAC clones described here were obtained indirectional chromosome walking experiments using D12S8 and the CHOP geneas initial starting points, the chromosome 12 breakpoint mapping resultspresented here confirm our previous claim. The FISH results obtainedwith the complete YAC insert of Y9091 as molecular probe were confirmedindependently in FISH studies using cosmid clones containing sequencescorresponding to various regions of the insert of this YAC clone. Thisis of importance, as the independent confirmatory results make it ratherunlikely that the split signals observed with the complete insert ofY9091 can be explained otherwise than by a factual splitting ofsequences represented in the YAC. The presence, for instance, of highlyrelated genetic sequences on both sides of a chromosome breakpoint couldeasily lead to erroneous conclusions if they were based solely on FISHresults of a YAC insert. Finally, our mapping studies have alsoestablished conclusively the chromosomal orientation of the long-rangerestriction map we have generated in these studies. This orientation wasalready predicted on the basis of two-color FISH studies (unpublishedobservations).

The FISH studies, described here, enabled us to map the chromosome 12breakpoint in Ad-312/SV40 cells to the 190-kbp DNA interval between theestablished STSs RM48 and RM69. However, the breakpoint region can benarrowed somewhat further on the basis of the following. The fact thatY4854 was shown to span the breakpoint indicates that at least aconsiderable part of the telomeric half of this YAC clone must mapdistally to the breakpoint. Precisely how much remains to beestablished. On the other side, STS RM69 appeared to be located in aboutthe middle of the DNA insert of cosmid clone cRM69, suggesting that thebreakpoint is close to 25 kbp distally to RM69. Moreover, cRM69 appearedto lack RM110(data not shown) and, as cRM110 was found proximally to thechromosome 12 breakpoint in Ad-312/SV40 cells, the breakpoint should beeven further distal to RM69 than the earlier-mentioned 25 kbp.Altogether, this narrows the chromosome 12 breakpoint region to a DNAinterval, which must be considerably smaller than 165 kbp. Furtherpinpointing of the breakpoint will allow us to molecularly clone thechromosome 12 breakpoint and to characterize the genetic sequences inthe breakpoint junction region, which might lead to the identificationof pathogenetically relevant sequences. Identification of the genespresent in the DNA inserts of YAC clones Y4854 and Y9091, viasequencing, direct hybridization, direct selection or exon-trapping,might constitute a useful alternative approach for identifying the genein this region of the long arm of chromosome 12 that might bepathogenetically critical for pleomorphic salivary gland adenomatumorigenesis.

The observation that the chromosome 12 breakpoints in other pleomorphicsalivary gland adenomas are located in a remote and more proximal regionon the long arm of chromosome 12 is of interest. It could imply that thechromosome 12 breakpoints in pleomorphic salivary gland adenomas aredispersed over a relatively large DNA region of the long arm ofchromosome 12, reminiscent to the 11q13 breakpoints in B-cellmalignancies [22]. Elucidation of the precise location of the chromosome12 breakpoints in the other pleomorphic salivary gland adenoma celllines could shed more light on this matter. On the other hand, it couldpoint towards alternative sequences on the long arm of chromosome 12between D12S8 and the CHOP gene that might be of importance, presumablyfor growth regulation in pleomorphic salivary gland adenoma. The factthat the chromosome 12 breakpoint region described here has sofar beenfound only in the Ad-312/SV40 cell line makes it necessary to analyze alarger number of salivary gland adenomas with chromosome 12q13-q15aberrations to assess the potential relevance for tumorigenesis of thechromosome 12 sequences affected in the studied cell line. If more caseswith aberrations in this particular region of chromosome 12 can befound, it would be of interest to find out whether these tumors form aclinical subgroup. Finally, chromosome translocations involving regionq13-q15 of human chromosome 12 have been reported for a variety of othersolid tumors: benign adipose tissue tumors, uterine leiomyoma,rhabdomyosarcoma, hemangiopericytoma, clear-cell sarcoma, chondromatoustumors, and hamartoma of the lung. Whether or not the chromosome 12breakpoints in some of these tumors map within the same region as thatof Ad-312/SV40 remains to be established. The YAC and cosmid clonesdescribed in this report constitute useful tools to investigate this.

The availability of a copy of the first-generation CEPH YAC library[10]and a copy of the arrayed chromosome 12-specific cosmid library(LLNL12NC01) [11] is greatly acknowledged. The cosmid library wasconstructed as part of the National Laboratory Gene Library Projectunder the auspices of the U.S. DOE by LLNL under contract No.W-7405-Eng-48. The authors acknowledge the excellent technicalassistance of M. Dehaen, C. Huysmans, E. Meyen, K. Meyer-Bolte, and M.Willems and would like to thank M. Leys for art work. This work wassupported in part by the EC through Biomed 1 program “MolecularCytogenetics of Solid Tumours”, the “Geconcerteerde onderzoekacties1992-1996”, the “Association Luxembourgeoise contre le Cancer”, theNational Fund for Scientific Research (NFWO; Kom op tegen Kanker), the“ASLK-programma voor Kankeronderzoek”, the “Schwerpunktprogramm:Molekulare und Klassische Tumorcytogenetik” of the DeutscheForschungsgemeinschaft, and the Tönjes-Vagt Stiftung. This text presentsresults of the Belgian programme on Interuniversity Poles of Attractioninitiated by the Belgian State, Prime Minister's Office, Science PolicyProgramming. The scientific responsibility is assumed by its authors. J.W. M. Geurts is an “Aspirant” of the National Fund for ScientificResearch (NFWO; Kom op tegen Kanker).

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20. Kools P F J, Roebroek A J M, Van de Velde H J K, Marynen P,Bullerdiek J, Van de Ven W J M (1993): Regional mapping of the human NSPgene to chromosome 14q21-q22 by fluorescence in situ hybridization.Cytogenet Cell Genet 66:48-50.

21. Looijenga L H J, Smit V T H B M, Wessels J W, Mollevanger P,Oosterhuis J W, Cornelisse C J (1990): Localization and polymorphism ofa chromosome 12-specific a satellite DNA sequence. Cytogenet Cell Genet53: 216-218.

22. Raynaud S D, Bekri S, Leroux D, Grosgeorge J, Klein B, Bastard C,Gaudray P, Simon M P (1993): Expanded range of 11q13 breakpoints withdiffering patterns of cyclin DI expression in B-cell malignancies. GenesChrom Cancer 8: 80-87.

Legends of Figures of Annex 2

FIG. 1. Composite physical map of the overlapping DNA inserts of YACclones Y4854 and Y9091. Sizes of the DNA inserts are indicated. Therelative positions of the YAC clones are represented by bars below thelong range physical map. Sequence-tagged sites (STSs) corresponding toend-clones of YACs, including YACs not shown here, are indicated byboxed RM codes above the restriction map. STSs obtained frominter-Alu-PCR products are given below the restriction map and the DNAregions to which they have been mapped are marked by arrows. B: BssHII;M: MluI; P: PvuI; Sf: SfiI. A polymorphic MluI site is marked by anasterisk.

FIG. 2. A) Mapping of cosmid clone cPK12qter to the telomeric region ofthe long arm of chromosome 12. Centromere 12-specific probe pα12H8 wasused to establish the identity of chromosome 12. FISH analysis wasperformed on metaphase chromosomes of control human lymphocytes.Hybridization signals of cPK12qter are marked with small arrowheads,those of the centromere 12-specific probe with asterisks. B, C) FISHanalysis of metaphase chromosomes of Ad-312/SV40 cells using DNA of YACclone Y4854 (B) or Y9091 (C) as molecular probe in combination withcosmid clone cPK12qter as reference marker. Hybridization signals of theYAC clones on chromosome 12 are indicated by large arrowheads; those onder(1) by large arrows, and those on der(12) by small arrows,respectively. The hybridization signals of cosmid clone cPK12qter areindicated by small arrowheads.

FIG. 3. FISH analysis of metaphase chromosomes of Ad-312/SV40 cellsusing DNA of cosmid clone cRM69 (A) or cRM111 (B) as molecular probe incombination with cosmid clone cPK12qter as reference marker. Theposition of the hybridization signals of cPK12qter are indicated bysmall arrowheads. In (A), the position of the hybridization signal ofcRM69 on normal chromosome 12 is indicated by a large arrowhead, andthat on der(12) with a small arrow. In (B), the position of thehybridization signal of cRM111 on normal chromosome 12 is indicated by alarge arrowhead, and that on der(1) with a large arrow.

FIG. 4. Schematic representation of FISH mapping data obtained for thesix pleomorphic salivary gland adenoma cell lines tested in this study.The specific chromosome 12 aberrations in the various cell lines aregiven. Cosmid clones which were used as probes in the FISH mappingstudies correspond to sequence-tagged sites obtained from overlappingYAC clones. Individual FISH experiments are indicated by dots. Cosmidclones were named after the acronyms of the STSs, as shown in the boxes,and the relative order of these is as presented. The DNA intervalbetween RM90 and RM103 is estimated to be about 1.3 Mb. Insert:Schematic representation of the G-banded derivative chromosomes der(1)and der(12) of the Ad-312/SV40 cell line, which carries a t(1;12)(p22;g15). The positions of the chromosome 12 breakpoints ofAd-248/SV40, Ad-263/SV40, Ad-295/SV40, Ad-302/SV40, and Ad-366/SV40 aredistal to RM103 as indicated by the arrow.

164 34 base pairs nucleic acid single linear Other 1 AAGGATCCGTCGACATCTTT TTTTTTTTTT TTTT 34 29 base pairs nucleic acid single linearOther 2 CUACUACUAC UAAAGGATCC GTCGACATC 29 18 base pairs nucleic acidsingle linear Other 3 CTTCAGCCCA GGGACAAC 18 18 base pairs nucleic acidsingle linear Other 4 CAAGAGGCAG ACCTAGGA 18 23 base pairs nucleic acidsingle linear Other 5 AACAATGCAA CTTTTAATTA CTG 23 30 base pairs nucleicacid single linear Other 6 CAUCAUCAUC AUCGCCTCAG AAGAGAGGAC 30 30 basepairs nucleic acid single linear Other 7 CAUCAUCAUC AUGTTCAGAAGAAGCCTGCT 30 35 base pairs nucleic acid single linear Other 8CAUCAUCAUC AUTTGATCTG ATAAGCAAGA GTGGG 35 21 base pairs nucleic acidsingle linear 9 CTCCAAGACA GGCCTCTGAT G 21 22 base pairs nucleic acidsingle linear 10 ACCACAGGTC CCCTTCAAAC TA 22 18 base pairs nucleic acidsingle linear 11 TCCTCCTGAG CAGGCTTC 18 18 base pairs nucleic acidsingle linear 12 CTTCAGCCCA GGGACAAC 18 18 base pairs nucleic acidsingle linear 13 CGCCTCAGAA GAGAGGAC 18 25 amino acids amino acid singlelinear None 14 Ala Arg Gly Glu Gly Ala Gly Gln Pro Ser Thr Ser Ala GlnGly Gln 1 5 10 15 Pro Ala Ala Pro Ala Pro Gln Lys Arg 20 25 17 aminoacids amino acid single linear None 15 Ser Pro Ser Lys Ala Ala Gln LysLys Ala Glu Ala Thr Gly Glu Lys 1 5 10 15 Arg 17 amino acids amino acidsingle linear None 16 Pro Arg Lys Trp Pro Gln Gln Val Val Gln Lys LysPro Ala Gln Glu 1 5 10 15 Glu 20 base pairs nucleic acid single linear17 TGGGACTAAC GGATTTTCAA 20 20 base pairs nucleic acid single linear 18TGTGGTTCAT TCATGCATTA 20 20 base pairs nucleic acid single linear 19TCCATCATCA TCTCAAAACA 20 22 base pairs nucleic acid single linear 20CTCTACCAAA TGGAATAAAC AG 22 20 base pairs nucleic acid single linear 21GCAGCTCAGG CTCCTTCCCA 20 20 base pairs nucleic acid single linear 22TGGCTTCCTG AAACGCGAGA 20 20 base pairs nucleic acid single linear 23TCTCCACTGC TTCCATTCAC 20 20 base pairs nucleic acid single linear 24ACACAAAACC ACTGGGGTCT 20 20 base pairs nucleic acid single linear 25CAGCTTTGGA ATCAGTGAGG 20 20 base pairs nucleic acid single linear 26CCTGGGGAAG AGGAGTAAAG 20 18 base pairs nucleic acid single linear 27GAGCTTCCTA TCTCATCC 18 18 base pairs nucleic acid single linear 28ATGCTTGTGT GTGAGTGG 18 18 base pairs nucleic acid single linear 29TTTGCTAAGC TAGGTGCC 18 18 base pairs nucleic acid single linear 30AGCTTCAAGA CCCATGAG 18 18 base pairs nucleic acid single linear 31CAGTTCTGAG ACTGCTTG 18 18 base pairs nucleic acid single linear 32TAATAGCAGG GACTCAGC 18 22 base pairs nucleic acid single linear 33CTTGTCTCAT TCTTTTAAAG GG 22 20 base pairs nucleic acid single linear 34CACCCCTTTT TAGATCCTAC 20 20 base pairs nucleic acid single linear 35GAATGTTCAT CACAGTGCTG 20 20 base pairs nucleic acid single linear 36AATGTGAGGT TCTGCTGAAG 20 20 base pairs nucleic acid single linear 37TTCTCATGGG GTAAGGACAG 20 22 base pairs nucleic acid single linear 38AAAGCTGCTT ATATAGGGAA TC 22 22 base pairs nucleic acid single linear 39CCTTGGCTTA GATATGATAC AC 22 22 base pairs nucleic acid single linear 40GCTCTTCAGA AATATCCTAT GG 22 20 base pairs nucleic acid single linear 41CCTTAGCAGT TGCTTGTCTG 20 20 base pairs nucleic acid single linear 42TCGTCACAGG ACATAGTCAC 20 23 base pairs nucleic acid single linear 43TCTATGGTAT GTTATACAAG ATG 23 19 base pairs nucleic acid single linear 44CAGTGAGATC CTGTCTCTA 19 24 base pairs nucleic acid single linear 45TCTGTGATGT TTTAAGCCAC TTAG 24 20 base pairs nucleic acid single linear46 AATTCTGTGT CCCTGCCACC 20 20 base pairs nucleic acid single linear 47ATTCTTCCTC ACCTCCCACC 20 20 base pairs nucleic acid single linear 48AATCTGCAGA GAGGTCCAGC 20 20 base pairs nucleic acid single linear 49AATTCTCCAT CTGGGCCTGG 20 20 base pairs nucleic acid single linear 50GAACGCTAAG CATGTGGGAG 20 20 base pairs nucleic acid single linear 51CTCCAACCAT GGTCCAAAAC 20 20 base pairs nucleic acid single linear 52GACCTCCAGT GGCTCTTTAG 20 20 base pairs nucleic acid single linear 53ACCATCAGAT CTGGCACTGA 20 20 base pairs nucleic acid single linear 54TTACATTGGA GCTGTCATGC 20 20 base pairs nucleic acid single linear 55TCCAGGACAT CCTGAAAATG 20 20 base pairs nucleic acid single linear 56AGTATCCTGC ACTTCTGCAG 20 21 base pairs nucleic acid single linear 57GATGAACTCT GAGGTGCCTT C 21 20 base pairs nucleic acid single linear 58TCAAACCCAG CTTTGACTCC 20 20 base pairs nucleic acid single linear 59GTCTTCAAAA CGCTTTCCTG 20 20 base pairs nucleic acid single linear 60TGGTTTGCAT AATGGTGATG 20 20 base pairs nucleic acid single linear 61TACACTACTC TGCAGCACAC 20 20 base pairs nucleic acid single linear 62TCTGAGTCAA TCACATGTCC 20 20 base pairs nucleic acid single linear 63CTCCCCAGAT GATCTCTTTC 20 20 base pairs nucleic acid single linear 64CGGTAGGAAA TAAAGGAGAG 20 20 base pairs nucleic acid single linear 65TATTTACTAG CTGGCCTTGG 20 20 base pairs nucleic acid single linear 66CATCTCAGGC ACACACAATG 20 20 base pairs nucleic acid single linear 67ATTCAGAGAA GTGGCCAAGT 20 20 base pairs nucleic acid single linear 68GGGATAGGTC TTCTGCAATC 20 20 base pairs nucleic acid single linear 69TCCAACAATA CTGAGTGACC 20 20 base pairs nucleic acid single linear 70TCCATTTCAC TGTAGCACTG 20 20 base pairs nucleic acid single linear 71GTAATCAACC ATTCCCCTGA 20 20 base pairs nucleic acid single linear 72AAAATAGCTG GTATGGTGGC 20 20 base pairs nucleic acid single linear 73ACTGCTCTAG TTTTCAAGGA 20 20 base pairs nucleic acid single linear 74AATTTACCTG ACAGTTTCCT 20 20 base pairs nucleic acid single linear 75GCATTTGACG TCCAATATTG 20 20 base pairs nucleic acid single linear 76ATTCCATTGG CTAACACAAG 20 20 base pairs nucleic acid single linear 77GCAAAACTTT GACTGAAACG 20 20 base pairs nucleic acid single linear 78CACAGAGTAT CGCACTGCAT 20 20 base pairs nucleic acid single linear 79AAGAGATTTC CCATGTTGTG 20 20 base pairs nucleic acid single linear 80CTAGTGCCTT CACAAGAACC 20 20 base pairs nucleic acid single linear 81AATTCTTGAG GGGTTCACTG 20 20 base pairs nucleic acid single linear 82TCCACACTGA GAGCTTTTCA 20 20 base pairs nucleic acid single linear 83GTGGTTCTGT ACAGCAGTGG 20 20 base pairs nucleic acid single linear 84TGAGAAAATG TCTGCCAAAT 20 20 base pairs nucleic acid single linear 85GCTCTACCAG GCATACAGTG 20 20 base pairs nucleic acid single linear 86ATTCCTAGCA TCTTTTCACG 20 20 base pairs nucleic acid single linear 87ATATGCATTA GGCTCAACCC 20 20 base pairs nucleic acid single linear 88ATCCCACAGG TCAACATGAC 20 25 base pairs nucleic acid single linear 89ATCCTTACAT TTCCAGTGGC ATTCA 25 23 base pairs nucleic acid single linear90 CCCAGAAGAC CCACATTCCT CAT 23 25 base pairs nucleic acid single linear91 TTTTAAGTTT CTCCAGGGAG GAGAC 25 25 base pairs nucleic acid singlelinear 92 AATAGGCTCT TTGGAAAGCT GGAGT 25 26 base pairs nucleic acidsingle linear 93 TCTCAGCTTA ATCCAAGAAG GACTTC 26 26 base pairs nucleicacid single linear 94 GGCATATTCC TCAACAATTT ATGCTT 26 25 base pairsnucleic acid single linear 95 TGGAGAAGCT ATGGTGCTTC CTATG 25 28 basepairs nucleic acid single linear 96 TGACAAATAG GTGAGGGAAA GTTGTTAT 28 20base pairs nucleic acid single linear 97 TCACACGCTG AATCAATCTT 20 20base pairs nucleic acid single linear 98 CAGCAGCTGA TACAAGCTTT 20 20base pairs nucleic acid single linear 99 TGTTTTCTTT CCCGATAGGT 20 20base pairs nucleic acid single linear 100 CTGGGATGCT CTTCGACCTC 20 22base pairs nucleic acid single linear 101 CCATCCAACA TCTTAAATGG AC 22 23base pairs nucleic acid single linear 102 CAGCTGCAAA CTCTAGGACT ATT 2310 base pairs nucleic acid single linear 103 TAGGAAATGG 10 10 base pairsnucleic acid single linear 104 GTGAGTAATA 10 10 base pairs nucleic acidsingle linear 105 AATACTCTGG 10 10 base pairs nucleic acid single linear106 CCTACTATTG 10 10 base pairs nucleic acid single linear 107GGAAGTGTGA 10 10 base pairs nucleic acid single linear 108 AACACAGGAC 1010 base pairs nucleic acid single linear 109 GTTTAATATT 10 10 base pairsnucleic acid single linear 110 AAGAAGGCAG 10 10 base pairs nucleic acidsingle linear 111 TAGGAGGTAG 10 10 base pairs nucleic acid single linear112 GGTGGCCATT 10 10 base pairs nucleic acid single linear 113GACAATCTAC 10 10 base pairs nucleic acid single linear 114 GTACAGAAGA 1010 base pairs nucleic acid single linear 115 GGGCATTCAG 10 10 base pairsnucleic acid single linear 116 GCAGTCTGTA 10 10 base pairs nucleic acidsingle linear 117 TCTGTATCCT 10 10 base pairs nucleic acid single linear118 ACACACTTCC 10 10 base pairs nucleic acid single linear 119ATATTATGGA 10 10 base pairs nucleic acid single linear 120 GAGGAGTTTT 1010 base pairs nucleic acid single linear 121 TAACACAGGA 10 10 base pairsnucleic acid single linear 122 TTACCTGCTG 10 10 base pairs nucleic acidsingle linear 123 GCTGGAGTGC 10 10 base pairs nucleic acid single linear124 GTCTCCTCCC 10 10 base pairs nucleic acid single linear 125TTTNTCTCTT 10 10 base pairs nucleic acid single linear 126 AGTCCAAGAA 1010 base pairs nucleic acid single linear 127 CCAAACTCTG 10 10 base pairsnucleic acid single linear 128 CTCCAGAAAC 10 10 base pairs nucleic acidsingle linear 129 AACTTCTTGA 10 10 base pairs nucleic acid single linear130 GAATGTCAGA 10 10 base pairs nucleic acid single linear 131CCTGGAAGCT 10 10 base pairs nucleic acid single linear 132 ATGGAGTCTC 1010 base pairs nucleic acid single linear 133 TTCCAGATAC 10 10 base pairsnucleic acid single linear 134 GCCTGCTCAG 10 10 base pairs nucleic acidsingle linear 135 TATCCTTTCA 10 10 base pairs nucleic acid single linear136 TCTTTCCACT 10 10 base pairs nucleic acid single linear 137ATACCACTTA 10 10 base pairs nucleic acid single linear 138 TTGCCATGGT 1010 base pairs nucleic acid single linear 139 CACTTTCATC 10 10 base pairsnucleic acid single linear 140 ATAAGGACTA 10 10 base pairs nucleic acidsingle linear 141 NCTTGTNAGC 10 10 base pairs nucleic acid single linear142 GTAAGACATA 10 10 base pairs nucleic acid single linear 143GTCAATGTTG 10 10 base pairs nucleic acid single linear 144 TCCTGGTACC 1010 base pairs nucleic acid single linear 145 TCTTTCAGAT 10 10 base pairsnucleic acid single linear 146 AATTACCTCT 10 10 base pairs nucleic acidsingle linear 147 GACTGACTCA 10 10 base pairs nucleic acid single linear148 TATTCCTGAA 10 10 base pairs nucleic acid single linear 149GTCAATGTTG 10 10 base pairs nucleic acid single linear 150 AATTACCTCT 1010 base pairs nucleic acid single linear 151 GCTTTTTCAA 10 10 base pairsnucleic acid single linear 152 GTTAAGAAAC 10 10 base pairs nucleic acidsingle linear 153 GNCTGACTAC 10 10 base pairs nucleic acid single linear154 AAGTCAAGAG 10 10 base pairs nucleic acid single linear 155TTTTAAAACA 10 10 base pairs nucleic acid single linear 156 AATCTGAAAT 1010 base pairs nucleic acid single linear 157 ATATGGCAAG 10 10 base pairsnucleic acid single linear 158 TCAGGCATCA 10 10 base pairs nucleic acidsingle linear 159 TAGAGATTAG 10 1033 base pairs nucleic acid singlelinear cDNA 160 CGCTTCAGAA GAGAGGACGC GGCCGCCCCA GGAAGCAGCA GCAAAAACCAACCGGTGAGC 60 CCTCTCCTAA GAGACCCAGG GGAAGACCCA AAGGCAGCAA AAACAAGAGTCCCTCTAAAG 120 CAGCTCAAGA GGAAGCAGAA GCCACTGAAG AAAAACGGCC AAGGGGCAGACCTAGGAAAT 180 GGGGTGGCCA TTCAGGGCAA CTGGGGCCTT CGTCAGTTGC CCCTTCATTCCGCCCAGAGG 240 ATGAGCTTGA GCACCTGACC AAAAAGATGC TGTATGACAT GGAAAATCCACCTGCTGACG 300 AATACTTTGG CCGCTGTGCT CGCTGTGGAG AAAACGTAGT TGGGGAAGGTACAGGATGCA 360 CTGCCATGGA TCAGGTCTTC CACGTGGATT GTTTTACCTG CATCATCTGCAACAACAAGC 420 TCCGAGGGCA GCCATTCTAT GCTGTGGAAA AGAAAGCATA CTGCGAGCCCTGCTACATTA 480 ATACTCTGGA GCAGTGCAAT GTGTGTTCCA AGCCCATCAT GGAGCGGATTCTCCGAGCCA 540 CCGGGAAGGC CTATCATCCT CACTGTTTCA CCTGCGTGAT GTGCCACCGCAGCCTGGATG 600 GGATCCCATT CACTGTGGAT GCTGGCGGGC TCATTCACTG CATTGAGGACTTCCACAAGA 660 AATTTGCCCC GCGGTGTTCT GTGTGCAAGG AGCCTATTAT GCCAGCCCCGGGCCAGGAGG 720 AGACTGTCCG TATTGTGGCT TTGGATCGAG ATTTCCATGT TCACTGCTACCGATGCGAGG 780 ATTGCGGTGG TCTCCTGTCT GAAGGAGATA ACCAAGGCTG CTACCCCTTGGATGGGCACA 840 TCCTCTGCAA GACCTGCAAC TCTGCCCGCA TCAGGGTGTT GACCGCCAAGGCGAGCACTG 900 ACCTTTAGAT TCAGTCACCT GTTCAGCCGG CACTGAGAAG AACGAACACAAGAAAAAGAT 960 AAGAAATACT AGAGTAAAGG CCATCAAACT ACGCGAAAAA AAAAAAAAAAAAAAAAGATG 1020 TCGACGGATC CTT 1033 343 amino acids amino acid singlelinear None 161 Leu Gln Lys Arg Gly Arg Gly Arg Pro Arg Lys Gln Gln GlnLys Pro 1 5 10 15 Thr Gly Glu Pro Ser Pro Lys Arg Pro Arg Gly Arg ProLys Gly Ser 20 25 30 Lys Asn Lys Ser Pro Ser Lys Ala Ala Gln Glu Glu AlaGlu Ala Thr 35 40 45 Glu Glu Lys Arg Pro Arg Gly Arg Pro Arg Lys Trp GlyGly His Ser 50 55 60 Gly Gln Leu Gly Pro Ser Ser Val Ala Pro Ser Phe ArgPro Glu Asp 65 70 75 80 Glu Leu Glu His Leu Thr Lys Lys Met Leu Tyr AspMet Glu Asn Pro 85 90 95 Pro Ala Asp Glu Tyr Phe Gly Arg Cys Ala Arg CysGly Glu Asn Val 100 105 110 Val Gly Glu Gly Thr Gly Cys Thr Ala Met AspGln Val Phe His Val 115 120 125 Asp Cys Phe Thr Cys Ile Ile Cys Asn AsnLys Leu Arg Gly Gln Pro 130 135 140 Phe Tyr Ala Val Glu Lys Lys Ala TyrCys Glu Pro Cys Tyr Ile Asn 145 150 155 160 Thr Leu Glu Gln Cys Asn ValCys Ser Lys Pro Ile Met Glu Arg Ile 165 170 175 Leu Arg Ala Thr Gly LysAla Tyr His Pro His Cys Phe Thr Cys Val 180 185 190 Met Cys His Arg SerLeu Asp Gly Ile Pro Phe Thr Val Asp Ala Gly 195 200 205 Gly Leu Ile HisCys Ile Glu Asp Phe His Lys Lys Phe Ala Pro Arg 210 215 220 Cys Ser ValCys Lys Glu Pro Ile Met Pro Ala Pro Gly Gln Glu Glu 225 230 235 240 ThrVal Arg Ile Val Ala Leu Asp Arg Asp Phe His Val His Cys Tyr 245 250 255Arg Cys Glu Asp Cys Gly Gly Leu Leu Ser Glu Gly Asp Asn Gln Gly 260 265270 Cys Tyr Pro Leu Asp Gly His Ile Leu Cys Lys Thr Cys Asn Ser Ala 275280 285 Arg Ile Arg Val Leu Thr Ala Lys Ala Ser Thr Asp Leu Xaa Ile Gln290 295 300 Ser Pro Val Gln Pro Ala Leu Arg Arg Thr Asn Thr Arg Lys ArgXaa 305 310 315 320 Glu Ile Leu Glu Xaa Arg Pro Ser Asn Tyr Ala Lys LysLys Lys Lys 325 330 335 Lys Lys Asp Val Asp Gly Ser 340 4323 base pairsnucleic acid single linear cDNA 162 GTCACTTTTA TTTGGGGGTG TGGACAGCTGCTTTCCCAGG GGAGTACTTC TTACAGTGGG 60 ATTTCAAGAC AAGATCGGCC TGAAGAAAAATTATATTTGT ATATTTTTTA AAAAGTGGGA 120 ACTTTGAGGC TCAGAGACAG AGCAGAAGACAGAACCTGGT CTTCTGATTC CCTGTGTTCT 180 GCTTTTTTCA TTGTTCCACT GGACGCTCATCAGAGGGAAG ATCTTTTTCC TCAATTGATT 240 CCAACAATGT CTCACCCATC TTGGCTGCCACCCAAAAGCA CTGGTGAGCC CCTCGGCCAT 300 GTGCCTGCAC GGATGGAGAC CACCCATTCCTTTGGGAACC CCAGCATTTC AGTGTCTACA 360 CAACAGCCAC CCAAAAAGTT TGCCCCGGTAGTTGCTCCAA AACCTAAGTA CAACCCATAC 420 AAACAACCTG GAGGTGAGGG TGATTTTCTTCCACCCCCAC CTCCACCTCT AGATGATTCC 480 AGTGCCCTTC CATCTATCTC TGGAAACTTTCCTCCTCCAC CACCTCTTGA TGAAGAGGCT 540 TTCAAAGTAC AGGGGAATCC CGGAGGCAAGACACTTGAGG AGAGGCGCTC CAGCCTGGAC 600 GCTGAGATTG ACTCCTTGAC CAGCATCTTGGCTGACCTTG AGTGCAGCTC CCCCTATAAG 660 CCTCGGCCTC CACAGAGCTC CACTGGTTCAACAGCCTCTC CTCCAGTTTC GACCCCAGTC 720 ACAGGACACA AGAGAATGGT CATCCCGAACCAACCCCCTC TAACAGCAAC CAAGAAGTCT 780 ACATTGAAAC CACAGCCTGC ACCCCAGGCTGGACCCATCC CTGTGGCTCC AATCGGAACA 840 CTCAAACCCC AGCCTCAGCC AGTCCCAGCCTCCTACACCA CGGCCTCCAC TTCTTCAAGG 900 CCTACCTTTA ATGTGCAGGT GAAGTCAGCCCAGCCCAGCC CTCATTATAT GGCTGCCCCT 960 TCATCAGGAC AAATTTATGG CTCAGGGCCCCAGGGCTATA ACACTCAGCC AGTTCCTGTC 1020 TCTGGGCAGT GTCCACCTCC TTCAACACGGGGAGGCATGG ATTATGCCTA CATTCCACCA 1080 CCAGGACTTC AGCCGGAGCC TGGGTATGGGTATGCCCCCA ACCAGGGACG CTATTATGAA 1140 GGCTACTATG CAGCAGGGCC AGGCTATGGGGGCAGAAATG ACTCTGACCC TACCTATGGT 1200 CAACAAGGTC ACCCAAATAC CTGGAAACGGGAACCAGGGT ACACTCCTCC TGGAGCAGGG 1260 AACCAGAACC CTCCTGGGAT GTATCCAGTCACTGGTCCCA AGAAGACCTA TATCACAGAT 1320 CCTGTTTCAG CCCCCTGTGC GCCACCATTGCAGCCAAAGG GTGGCCATTC AGGGCAACTG 1380 GGGCCTTCGT CAGTTGCCCC TTCATTCCGCCCAGAGGATG AGCTTGAGCA CCTGACCAAA 1440 AAGATGCTGT ATGACATGGA AAATCCACCTGCTGACGAAT ACTTTGGCCG CTGTGCTCGC 1500 TGTGGAGAAA ACGTAGTTGG GGAAGGTACAGGATGCACTG CCATGGATCA GGTCTTCCAC 1560 GTGGATTGTT TTACCTGCAT CATCTGCAACAACAAGCTCC GAGGGCAGCC ATTCTATGCT 1620 GTGGAAAAGA AAGCATACTG CGAGCCCTGCTACATTAATA CTCTGGAGCA GTGCAATGTG 1680 TGTTCCAAGC CCATCATGGA GCGGATTCTCCGAGCCACCG GGAAGGCCTA TCATCCTCAC 1740 TGTTTCACCT GCGTGATGTG CCACCGCAGCCTGGATGGGA TCCCATTCAC TGTGGATGCT 1800 GGCGGGCTCA TTCACTGCAT TGAGGACTTCCACAAGAAAT TTGCCCCGCG GTGTTCTGTG 1860 TGCAAGGAGC CTATTATGCC AGCCCCGGGCCAGGAGGAGA CTGTCCGTAT TGTGGCTTTG 1920 GATCGAGATT TCCATGTTCA CTGCTACCGATGCGAGGATT GCGGTGGTCT CCTGTCTGAA 1980 GGAGATAACC AAGGCTGCTA CCCCTTGGATGGGCACATCC TCTGCAAGAC CTGCAACTCT 2040 GCCCGCATCA GGGTGTTGAC CGCCAAGGCGAGCACTGACC TTTAGATTCA GTCACCTGTT 2100 CAGCCGGCAC TGAGAAGAAC GAACACAAGAAAAAGATAAG AAATACTAGA GTAAAGGCCA 2160 TCAAACTACG CGATAGTCTC TGTTCTTCATCTGCTATTAA CCTTGCCTTA GAAACACATA 2220 AATTATGAGA TTTTTTTTTA AAAGTTGTTACCAAATACAC ATTTCACATT GAATCATGTA 2280 GGATCTTGAT GGGCCTTTGT TCCCAAGGACTTCCACATTT TTGCACAGAT TATGCTCCAT 2340 CCCTTCACTT CTGCATTCCT GTAACTTTTAATCCCTATGT TTGTCTCACT TTTCATCTGG 2400 TTGAATGGCT TTTCTTAGTG TGGTATTTGCTGTCACATAG TTTTTTCCTG GGTGAGTCTG 2460 CCAACTCACA GGTGCTTTTA GGCTTGAAATCTCCATCCTA TCATTTCCGT TTTGCCTGTG 2520 ACTGTAAAGA GTAGCCATTC TTTTCCCATGTATTGAAGAG GATATTCTTC TCTTGCTTTA 2580 TACTACTCAC GTCCTTGGGG AGGGAAATGCACAATTTTTT TTTGTTAGGC TGTAAAGAAT 2640 TTAAGCTGTA AATTACATAA GTTAGAACAAGCCCAAATTT AATTTGCAAC CATCAGAATT 2700 CAGAATCTAT AGTGACCAGT GATCAAGGCTAATTGGAAAA GAGTTATCGG CCCATAGCTA 2760 ATAAGTAGTG ACAGACAACC AAGCTTCAATATTTTTCTAA AGAAATTACA GGTGGGATAT 2820 GCTAGAAAAG GCATTTTGGG GTTATGTTTAAAAAAACATT ATTGTCCCAC AATATTACCT 2880 TAAGATTTTT CTTTTCCGCA CTACCTGAACATTGTAATAC AGACAAACTT GATTTCTTCT 2940 AGAAGATAAC ATTTTCAATA CTGTCCCACTTCTCATCTTA AAAATATTGT CATGTTTATT 3000 CTAATATCCA ACGCAACTAT CAAAATTGCCTTTTTCTCTA GAGGATGAAG GCTGTGAAAA 3060 AACCGTTCAA ATTCTCTTCT TTTTCTTTTTTATTACCAGG TCCATTTTGC CTGACAATTG 3120 CAAATCAGAG CATACAAAAT AAAACTGTGCAGTTTTGTTT GGTTTACTTT CAAAAGAGTA 3180 GAAAGCTTGA AAAGATTCTG AAACCACAGTTTCATTATTC TCATAATCCT TCTGCAACTG 3240 AAATTACATA TTGCAGGAGA CATTTTCATATCATCAATCT GACATTTACA CCACACTTTC 3300 AAAGACAATC ACTGAAACAA AAATTGTCTTTATGAGCTAA AAATATGCAG AATCTCTGCC 3360 TAGAATCTTT ATTCAAACTT TTATTAGCCAGTGAAACACT TGCTTGCCAA CTGCCAAGCC 3420 ATACTTATTA AGTTCGAACA TGTTTCACTTAAGGAGAGAC ACCTAGCTTA GTCATGGCAA 3480 GTTGCCATTT TGTAAACTAA GGATTTTGGACTGAGATTTC TTAAATCTTT CTTCAAATCT 3540 CCCACAAGTA TATACTTTTA AATTATGGAGTATTTTAAGT CTACAAAAAG GTATAAATAA 3600 TAATATAATG AATTCCTATA TACCTAATACCCAGTTTAAG ACACCAAATA TAACAAGTAT 3660 AATTACATCC TCCAATGTAC CGTTTCCTTATTCCACAGAT ATCTTTTTCA TTATTGTGAA 3720 GTGATGTTCA GATTTCTAGT TTTTTTTTCTAGTTTTTAAT TTTAACATCA GAACTGAAAT 3780 AAAAAATTAT GGATACGTGT TTTGAATTGCAAACTATTCC TCAGGAATTC CAATTAAATT 3840 TATTTTACTT GAATAGGAAT GATCATAAAAGTGATTCTTT TTTTGTGACT AGAAATTCTT 3900 AAGCCGATGG TCACTATAGC TCATCCTTAATGTATGGCTC ATTTGCTTTT GTCACTAAAC 3960 GGTTTTGTGT TAGAACCACC AAAATTATAGCTTTTAAGAG CTTCCTTTGA CCACTGTCTT 4020 TTTCTTACCC TACTTCTCTT ATCTTTGATCGTATATTTCT CATAATGTGA AATATGATGA 4080 GATTCACTTA GGGGCAGCAT GTTAGTTTTGGGAGGCAATG TCAACTGTGT CTCTGAATTC 4140 CTGTCTTCCA AATTGAAGCC AGACCATGCTGATGACCTCA AGTAGCACTG ACTATTTGAC 4200 AATAGGGCTG ATAATGTAAT CGGCTTGAATTTTGACTTAG TAACTTTTTA TGTAATACTT 4260 TCGGAGAAAT TCTCTTTAGG ACAAAGCAGAGAGTCCAATT TATTGAGGGA TAGATTGTAT 4320 CTC 4323 612 amino acids aminoacid single linear 163 Met Ser His Pro Ser Trp Leu Pro Pro Lys Ser ThrGly Glu Pro Leu 1 5 10 15 Gly His Val Pro Ala Arg Met Glu Thr Thr HisSer Phe Gly Asn Pro 20 25 30 Ser Ile Ser Val Ser Thr Gln Gln Pro Pro LysLys Phe Ala Pro Val 35 40 45 Val Ala Pro Lys Pro Lys Tyr Asn Pro Tyr LysGln Pro Gly Gly Glu 50 55 60 Gly Asp Phe Leu Pro Pro Pro Pro Pro Pro LeuAsp Asp Ser Ser Ala 65 70 75 80 Leu Pro Ser Ile Ser Gly Asn Phe Pro ProPro Pro Pro Leu Asp Glu 85 90 95 Glu Ala Phe Lys Val Gln Gly Asn Pro GlyGly Lys Thr Leu Glu Glu 100 105 110 Arg Arg Ser Ser Leu Asp Ala Glu IleAsp Ser Leu Thr Ser Ile Leu 115 120 125 Ala Asp Leu Glu Cys Ser Ser ProTyr Lys Pro Arg Pro Pro Gln Ser 130 135 140 Ser Thr Gly Ser Thr Ala SerPro Pro Val Ser Thr Pro Val Thr Gly 145 150 155 160 His Lys Arg Met ValIle Pro Asn Gln Pro Pro Leu Thr Ala Thr Lys 165 170 175 Lys Ser Thr LeuLys Pro Gln Pro Ala Pro Gln Ala Gly Pro Ile Pro 180 185 190 Val Ala ProIle Gly Thr Leu Lys Pro Gln Pro Gln Pro Val Pro Ala 195 200 205 Ser TyrThr Thr Ala Ser Thr Ser Ser Arg Pro Thr Phe Asn Val Gln 210 215 220 ValLys Ser Ala Gln Pro Ser Pro His Tyr Met Ala Ala Pro Ser Ser 225 230 235240 Gly Gln Ile Tyr Gly Ser Gly Pro Gln Gly Tyr Asn Thr Gln Pro Val 245250 255 Pro Val Ser Gly Gln Cys Pro Pro Pro Ser Thr Arg Gly Gly Met Asp260 265 270 Tyr Ala Tyr Ile Pro Pro Pro Gly Leu Gln Pro Glu Pro Gly TyrGly 275 280 285 Tyr Ala Pro Asn Gln Gly Arg Tyr Tyr Glu Gly Tyr Tyr AlaAla Gly 290 295 300 Pro Gly Tyr Gly Gly Arg Asn Asp Ser Asp Pro Thr TyrGly Gln Gln 305 310 315 320 Gly His Pro Asn Thr Trp Lys Arg Glu Pro GlyTyr Thr Pro Pro Gly 325 330 335 Ala Gly Asn Gln Asn Pro Pro Gly Met TyrPro Val Thr Gly Pro Lys 340 345 350 Lys Thr Tyr Thr Thr Asp Pro Val SerAla Pro Cys Ala Pro Pro Leu 355 360 365 Gln Pro Lys Gly Gly His Ser GlyGln Leu Gly Pro Ser Ser Val Ala 370 375 380 Pro Ser Phe Arg Pro Glu AspGlu Leu Glu His Leu Thr Lys Lys Met 385 390 395 400 Leu Tyr Asp Met GluAsn Pro Pro Ala Asp Glu Tyr Phe Gly Arg Cys 405 410 415 Ala Arg Cys GlyGlu Asn Val Val Gly Glu Gly Thr Gly Cys Thr Ala 420 425 430 Met Asp GlnVal Phe His Val Asp Cys Phe Thr Cys Ile Ile Cys Asn 435 440 445 Asn LysLeu Arg Gly Gln Pro Phe Tyr Ala Val Glu Lys Lys Ala Tyr 450 455 460 CysGlu Pro Cys Tyr Ile Asn Thr Leu Glu Gln Cys Asn Val Cys Ser 465 470 475480 Lys Pro Ile Met Glu Arg Ile Leu Arg Ala Thr Gly Lys Ala Tyr His 485490 495 Pro His Cys Phe Thr Cys Val Met Cys His Arg Ser Leu Asp Gly Ile500 505 510 Pro Phe Thr Val Asp Ala Gly Gly Leu Ile His Cys Ile Glu AspPhe 515 520 525 Glu Lys Lys Phe Ala Pro Arg Cys Ser Val Cys Lys Glu ProIle Met 530 535 540 Pro Ala Pro Gly Gln Glu Glu Thr Val Arg Ile Val AlaLeu Asp Arg 545 550 555 560 Asp Phe His Val His Cys Tyr Arg Cys Glu AspCys Gly Gly Leu Leu 565 570 575 Ser Glu Gly Asp Asn Gln Gly Cys Tyr ProLeu Asp Gly His Ile Leu 580 585 590 Cys Lys Thr Cys Asn Ser Ala Arg IleArg Val Leu Thr Ala Lys Ala 595 600 605 Ser Thr Asp Leu 610 4067 basepairs nucleic acid single linear cDNA 164 CTTGAATCTT GGGGCAGGAACTCAGAAAAC TTCCAGCCCG GGCAGCCCGC GTTTGGTGCA 60 AGACTCAGGA GCTAGCAGCCCGTCCCCCTC CGACTCTCCG GTGCCGTTGC TGCCTGCTCC 120 CGCCACCCTA GGAGGCGCGGTGCCACCCAC TACTCTGTCC TCTGCCTGTG CTCCGTGCCC 180 GACCCTATCC CGGCGGAGTCTCCCCATCCT CCTTTGCTTT CCGACTGCCC AAGGCACTTT 240 CAATCTCAAT CTCTTCTCTCTCTCTCTCTC TCTCTCTGTC TCTCTCTCTC TCTCTCTCTC 300 TCTCTCTCTC GCAGGGTGGGGGGAAGAGGA GGAGGAATTC TTTCCCCGCC TAACATTTCA 360 AGGGACCACA ATCACTCCAAGTCTCTTCCC TTTCCAAGCC GCTTCCGAAG TGCTCCCGGT 420 GCCCGCAACT CCTGATCCCAACCCGCGAGA GGAGCCTCTG CGACCTCAAA GCCTCTCTTC 480 CTTCTCCCTC GCTTCCCTCCTCCTCTTGCT ACCTCCACCT CCACCGCCAC CTCCACCTCC 540 GGCACCCACC CACCGCCGCCGCCGCCACCG GCAGCGCCTC CTCCTCTCCT CCTCCTCCTC 600 CCCTCTTCTC TTTTTGGCAGCCGCTGGACG TCCGGTGTTG ATGGTGGCAG CGGCGGCAGC 660 CTAAGCAACA GCAGCCCTCGCAGCCCGCCA GCTCGCGCTC GCCCCGCCGG CGTCCCCAGC 720 CCTATCACCT CATCTCCCGAAAGGTGCTGG GCAGCTCCGG GGCGGTCGAG GCGAAGCGGC 780 TGCAGCGGCG GTAGCGGCGGCGGGAGGCAG GATGAGCGCA CGCGGTGAGG GCGCGGGGCA 840 GCCGTCCACT TCAGCCCAGGGACAACCTGC CGCCCCAGCG CCTCAGAAGA GAGGACGCGG 900 CCGCCCCAGG AAGCAGCAGCAAGAACCAAC CGGTGAGCCC TCTCCTAAGA GACCCAGGGG 960 AAGACCCAAA GGCAGCAAAAACAAGAGTCC CTCTAAAGCA GCTCAAAAGA AAGCAGAAGC 1020 CACTGGAGAA AAACGGCCAAGAGGCAGACC TAGGAAATGG CCACAACAAG TTGTTCAGAA 1080 GAAGCCTGCT CAGGAGGAAACTGAAGAGAC ATCCTCACAA GAGTCTGCCG AAGAGGACTA 1140 GGGGGCGCAA CGTTCGATTTCTACCTCAGC AGCAGTTGGA TCTTTTGAAG GGAGAAGACA 1200 CTGCAGTGAC CACTTATTCTGTATTGCCAT GGTCTTTCCA CTTTCATCTG GGGTGGGGTG 1260 GGGTGGGGTG GGGGAGGGGGGGGTGGGGTG GGGAGAAATC ACATAACCTT AAAAAGGACT 1320 ATATTAATCA CCTTCTTTGTAATCCCTTCA CAGTCCCAGG TTTAGTGAAA AACTGCTGTA 1380 AACACAGGGG ACACAGCTTAACAATGCAAC TTTTAATTAC TGTTTTCTTT TTTCTTAACC 1440 TACTAATAGT TTGTTGATCTGATAAGCAAG AGTGGGCGGG TGAGAAAAAC CGAATTGGGT 1500 TTAGTCAATC ACTGCACTGCATGCAAACAA GAAACGTGTC ACACTTGTGA CGTCGGGCAT 1560 TCATATAGGA AGAACGCGGTGTGTAACACT GTGTACACCT CAAATACCAC CCCAACCCAC 1620 TCCCTGTAGT GAATCCTCTGTTTAGAACAC CAAAGATAAG GACTAGATAC TACTTTCTCT 1680 TTTTCGTATA ATCTTGTAGACACTTACTTG ATGATTTTTA ACTTTTTATT TCTAAATGAG 1740 ACGAAATGCT GATGTATCCTTTCATTCAGC TAACAAACTA GAAAAGGTTA TGTTCATTTT 1800 TCAAAAAGGG AAGTAAGCAAACAAATATTG CCAACTCTTC TATTTATGGA TATCACACAT 1860 ATCAGCAGGA GTAATAAATTTACTCACAGC ACTTGTTTTC AGGACAACAC TTCATTTTCA 1920 GGAAATCTAC TTCCTACAGAGCCAAAATGC CATTTAGCAA TAAATAACAC TTGTCAGCCT 1980 CAGAGCATTT AAGGAAACTAGACAAGTAAA ATTATCCTCT TTGTAATTTA ATGAAAAGGT 2040 ACAACAGAAT AATGCATGATGAACTCACCT AATTATGAGG TGGGAGGAGC GAAATCTAAA 2100 TTTCTTTTGC TATAGTTATACATCAATTTA AAAAGCAAAA AAAAAAAGGG GGGGGCAATC 2160 TCTCTCTGTG TCTTTCTCTCTCTCTCTCCC TCTCCCTCTC TCTTTTCATG TGTATCAGTT 2220 TCCATGAAAG ACCTGAATACCACTTACCTC AAATTAAGCA TATGTGTTAC TTCAAGTAAT 2280 ACGTTTTGAC ATAAGATGGTTGACCAAGGT GCTTTTCTTC GGCTTGAGTT CACCATCTCT 2340 TCATTCAAAC TGCACTTTTAGCCAGAGATG CAATATATCC CCACTACTCA ATACTACCTC 2400 TGAATGTTAC AACGAATTTACAGTCTAGTA CTTATTACAT GCTGCTATAC ACAAGCAATG 2460 CAAGAAAAAA ACTTACTGGGTAGGTGATTC TAATCATCTG CAGTTCTTTT TGTACACTTA 2520 ATTACAGTTA AAGAAGCAATCTCCTTACTG TGTTTCAGCA TGACTATGTA TTTTTCTATG 2580 TTTTTTTAAT TAAAAATTTTTAAAATACTT GTTTCAGCTT CTCTGCTAGA TTTCTACATT 2640 AACTTGAAAA TTTTTTAACCAAGTCGCTCC TAGGTTCTTA AGGATAATTT TCCTCAATCA 2700 CACTACACAT CACACAAGATTTGACTGTAA TATTTAAATA TTACCCTCCA AGTCTGTACC 2760 TCAAATGAAT TCTTTAAGGAGATGGACTAA TTGACTTGCA AAGACCTACC TCCAGACTTC 2820 AAAAGGAATG AACTTGTTACTTGCAGCATT CATTTGTTTT TTCAATGTTT GAAATAGTTC 2880 AAACTGCAGC TAACCCTAGTCAAAACTATT TTTGTAAAAG ACATTTGATA GAAAGGAACA 2940 CGTTTTTACA TACTTTTGCAAAATAAGTAA ATAATAAATA AAATAAAGCC AACCTTCAAA 3000 GAACTTGAAG CTTTGTAGGTGAGATGCAAC AAGCCCTGCT TTTGCATAAT GCAATCAAAA 3060 ATATGTGTTT TTAAGATTAGTTGAATATAA GAAAATGCTT GACAAATATT TTCATGTATT 3120 TTACACAAAT GTGATTTTTGTAATATGTCT CAACCAGATT TATTTTAAAC GCTTCTTATG 3180 TAGAGTTTTT ATGCCTTTCTCTCCTAGTGA GTGTGCTGAC TTTTTAACAT GGTATTATCA 3240 ACTGGGCCAG GAGGTAGTTTCTCATGACGG CTTTTGTCAG TATGGCTTTT AGTACTGAAG 3300 CCAAATGAAA CTCAAAACCATCTCTCTTCC AGCTGCTTCA GGGAGGTAGT TTCAAAGGCC 3360 ACATACCTCT CTGAGACTGGCAGATCGCTC ACTGTTGTGA ATCACCAAAG GAGCTATGGA 3420 GAGAATTAAA ACTCAACATTACTGTTAACT GTGCGTTAAA TAAGCAAATA AACAGTGGCT 3480 CATAAAAATA AAAGTCGCATTCCATATCTT TGGATGGGCC TTTTAGAAAC CTCATTGGCC 3540 AGCTCATAAA ATGGAAGCAATTGCTCATGT TGGCCAAACA TGGTGCACCG AGTGATTTCC 3600 ATCTCTGGTA AAGTTACACTTTTATTTCCT GTATGTTGTA CAATCAAAAC ACACTACTAC 3660 CTCTTAAGTC CCAGTATACCTCATTTTTCA TACTGAAAAA AAAAGCTTGT GGCCAATGGA 3720 ACAGTAAGAA CATCATAAAATTTTTATATA TATAGTTTAT TTTTGTGGGA GATAAATTTT 3780 ATAGGACTGT TCTTTGCTGTTGTTGGTCGC AGCTACATAA GACTGGACAT TTAACTTTTC 3840 TACCATTTCT GCAAGTTAGGTATGTTTGCA GGAGAAAAGT ATCAAGACGT TTAACTGCAG 3900 TTGACTTTCT CCCTGTTCCTTTGAGTGTCT TCTAACTTTA TTCTTTGTTC TTTATGTAGA 3960 ATTGCTGTCT ATGATTGTACTTTGAATCGC TTGCTTGTTG AAAATATTTC TCTAGTGTAT 4020 TATCACTGTC TGTTCTGCACAATAAACATA ACAGCCTCTG TGATCCC 4067

What is claimed is:
 1. An isolated nucleic acid, comprising thelipoma-preferred partner sequence depicted in FIG. 5 (SEQ ID NO: 162) ora completely complementary strand thereof.
 2. The isolated nucleic acidof claim 1 in which the nucleic acid is labeled for use as a probe. 3.An expression vector comprising the lipoma-preferred partner sequencedepicted in FIG. 5 (SEQ ID NO: 162).
 4. A host cell comprising anexpression vector according to claim 3.